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Full text of "Review of intramural research 1962"

R853 

•U5601 

1962 

c.3 






LIBRAKY 

OCT 1 4 1965 

mum msiiijiib oe 



NATIONAL INSTITUTES OF HEALTH 



Review 
of 

INTRAMURAL 

RESEARCH 

1962 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 



ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE 



library 

°a, Mary/ancf 2OQI4 



NATIONAL INSTITUTES OF HEALTH 



Review 
of 

INTRAMURAL 



RESEARCH 
1962 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 
National Institutes of Health, Bethesda, Maryland 20014 



,'60 / 



INSTITUTE RESEARCH DIRECTORS 

National Cancer Institute Charles G. Zubrod, M.D. 

Scientific Director 
Nathaniel I. Berlin, M.D. 

Clinical Director 

National Heart Institute Robert W. Berliner, M.D. 

Scientific Director 
Donald S. Fredrickson, M.D. 

Clinical Director 
National Institute of Arthritis 

and Metabolic Diseases Joseph E. Rail, M.D. 

Scientific Director 
Joseph J. Bunim, M.D. 

Clinical Director 
National Institute of Allergy 

and Infectious Diseases Dorland J. Davis, M.D. 

Scientific Director 
Vernon Knight, M.D. 

Clinical Director 

National Institute of Mental Health John C. Eberhart, M.D. 

Scientific Director 
Robert A. Cohen, M.D. 

Clinical Director 
National Institute of Neurological 

Diseases and Blindness Richard L. Masland, M.D. 

Acting Scientific Director 
Maitland Baldwin, M.D. 

Clinical Director 

National Institute of Dental Research Seymour Kreshover, M.D., D.D.S. 

Scientific Director 
Edward J. Driscoll, D.D.S. 

Clinical, Director 

Division of Biologics Standards Roderick Murray, M.D. 

Scientific Director 

G. Burroughs Mider, M.D. 

Director of Laboratories and Clinics 

Roger K. McDonald, M.D. 

Assistant to Director of Laboratories and Clinics 



m 



FOREWORD 

The National Institutes of Health utilizes extramural and intramural programs in 
fulfilling its mission of conducting and supporting research, research training, and 
related activities in the biomedical sciences. The extramural activity, administered from 
Bethesda, affects most biomedical research institutions in the United States, and is now 
reaching into areas throughout the world. The intramural operation is carried out in 
the laboratories and clinics in Bethesda and seeks primarily new information in biology 
and medicine. This volume — which, it is believed, serves a number of scientific and 
institutional needs — summarizes the work done and knowledge gained in NIH's own 
laboratory and clinical facilities during calendar 1962. The reports are essentially in 
the form prepared by their originators, with only the most general guidelines, and 
without appreciable editorial modification. The reader may thus savor the diversity 
of outlook and attitude prevailing among NIH scientists, while noting that such di- 
versity in no way inhibits pursuit of the common goal of understanding and mastering 
disease processes. 

NIH's intramural programs embrace challenging scientific opportunities and major 
social responsibilities. During years of rapid growth and change in both substance and 
dimension of the health-related sciences, it is a matter of pride that NIH continues to 
meet these opportunities and responsibilities with undiminished standards of excellence. 



>^#,tt\&4 9. SkeOrv\<rr>w 



James A. Shannon, M.D., 
Director, National Institutes of Health. 



CONTENTS 



Foreword 

National Cancer Institute 

Introduction 

Specific highlights 

Natural history of the disease 

Acute leukemia 

Multiple myeloma 

Head and neck cancer 

Cervical cancer 

Infection in surgery 

Theory of basal cell cancer 

Therapy 

Biochemical and physiological studies 

Immunological 

Steroid chemistry 

Pyrimidine metabolism 

Pharmacology 

Normal skin 

Lymphangiography 

White cells and platelets 

Carcinogenesis 

Metabolism service 

Amino acid transport 

Gamma globulin structure 

Gamma globulin metabolism 

Albumin metabolism 

Immunological studies 

Nucleic acid and pyrimidine metabolism. . 

Studies of erythropoietin 

Effect of metabolic rate on erythropoiesis, 

Means of measuring red cell lifespan 

Porphyrins 

Dermatology branch 

Epidermal growth and differentiation 

In psoriasis 

In normal hair root 

In basal cell tumor 

Mycosis f ungoides 

Endocrinology branch 

Medicine branch 

Remission induction 

Remission maintenance 

Cytogenetics 

Pharmacology and related studies 

Biochemical studies 

Radiation branch 

Surgery branch 

Intramural research program 

Introduction 



Page 
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Page 

Laboratory of biochemistry 23 

Cytochemistry section 23 

Nucleic acids section 24 

Nutrition and carcinogenesis section 25 

Protein chemistry section 26 

Tumor-host relations section 28 

Laboratory of biology 29 

Carcinogenesis 30 

Immunology 30 

Virology 31 

Cell physiology and nutrition 31 

Genetics 32 

Biochemical genetics 33 

Pathogenesis 33 

Drug resistance 34 

Laboratory of chenical pharmacology 34 

Endotoxins 34 

Cancer immunology 36 

Tumor-producing viruses 37 

Chemotherapy 38 

Tumors studied 38 

Mice employed 38 

Chemical agents examined 38 

Topics investigated 38 

Team work and collaborative work 38 

Laboratory of pathology 39 

Collaborative research 39 

Accumulation of data 40 

Natural history of cancer in man; applications 

to research animals 41 

Carcinogenesis 41 

Viral carcinogenesis studies 41 

Chemical carcinogenesis 42 

Transplantable tumors 42 

Miscellaneous 45 

Intracerebral localization of leukenic 

L1210 cells in mice 45 

Hyperbilirubinemia rats 45 

Aging process in collagenous and elastic 

connective tissue 45 

Classification of neoplasms 45 

Monograph on cytological characteristics 

of neoplasms 45 

Laboratory of physiology 45 

Laboratory of viral oncology 48 

National Heart Institute 55 

Introduction 55 

vn 



Mil 



CONTENTS 



Page 

Laboratory of biochemistry 55 

Section on enzymes 55 

Metabolism of heterocyclic compounds — 55 

Nicotinic acid dissimilation 55 

Riboflavin degradation 56 

Fatty acid synthesis 56 

Regulation of fatty acid biosynthesis 57 

One carbon metabolism 57 

Formate activation 57 

Oxidation of methylamine 57 

Carbon dioxide activation 58 

Methane fermentation 58 

Metabolism of amino acids 58 

Lysine degradation 58 

Cystathionine metabolism 59 

Thioalkyl transfer 60 

Permeation and intracellular concentration 

of purines 60 

Hydrogen activation 60 

Ethylene glycol metabolism 61 

Section on cellular physiology 61 

Structure of ribonuclease 61 

Structure of lysozyme 63 

Myosin structure and activity 63 

Lipopeptides in protein synthesis 64 

Cell transport of lipids 64 

Laboratory of chemical pharmacology 64 

Mobilization and utilization of metabolic sub- 
strates 64 

FFA mobilization in starved rats 65 

Control of basal metabolism 65 

Neurochemical transducer 65 

Drugs interfering with biochemical control 

mechanisms 67 

Pituitary-adrenal system 67 

Lipid transport 67 

Factors affect duration of drug action 67 

Enzymatic mechanisms of drug metabo- 
lism 67 

Distribution of drugs 67 

Activators and inhibitors of drug metabo- 
lism 67 

Action of desmethylimipramine 68 

Species differences in DMI metabolism-. 68 

Passage of substances across membranes 68 

Membranes within CNS 68 

Biliary excretion of drugs 68 

Penetration of drugs into cells 68 

Drugs and uptake of norepinephrine and 

5HT 69 

Development of new drugs 69 

Antidepressants 69 

Dopamine hydroxylase blockers 69 

Bretylium-like compounds 69 

Development of new methods of analysis 69 

Laboratory of technical development 69 

Gas chromatography 69 

Cumulative collection on anthracene 70 

Fraction collection on anthracene 70 



Page 
Laboratory of technical development — Continued 
Gas chromatography — Continued 

Ionization chamber 70 

Flow-through scintillation 70 

Tritium assay 70 

Determination of blood gases 71 

D.C. discharge detector 71 

Quantitative micro determination of lipids 
Application of gas chromatography meth- 
ods 71 

Ultra microanalysis of sodium and potas- 
sium 71 

Ultra microfreezing point depression ap- 
paratus 71 

Blood flow measurement 72 

Fast reaction methods 72 

Calorimetry of intact cell metabolism 72 

Photochemistry of the ATPase system 73 

Fluorescence and phosphorescence 73 

Measurement and intracellular localiza- 
tion of tetracycline 73 

Theoretical analysis of biological trans- 
port problems 74 

Laboratory of cardiovascular physiology 74 

Atrium 74 

Atrial fibrillation 74 

Timing of atrial systole 75 

Closure of mitral valve 75 

Homeometric autoregulation 75 

Dynamics of homeometric autoregulation. 75 
Potassium efflux during homeometric auto- 
regulation 76 

Myocardial metabolism 77 

Changing coronary blood flow and myo- 
cardial 2 consumption 77 

Digitalis and myocardial 2 consumption. 77 

Synchronicity of ventricular contraction 78 

Reflexes arising from the heart 78 

Renal function 78 

Extrinsic (autonomic) factors 78 

Intrinsic factors (autoregulation) 79 

Renal blood flow distribution 79 

Kallidin and renal function 80 

Catechol amines 80 

Norepinephrine refractoriness 80 

Action of ephedrine sulfate 80 

Laboratory of kidney and electrolyte metabolism. _ 80 

Renal physiology 80 

Micropuncture studies in the dog 80 

Nonelectrolytes and urine concentration.. 81 

Measurement of medullary blood flow 81 

Electrolyte and water transport 82 

Electrolyte fluxes in renal tubules of rabbit— 82 

Red cell ghosts 83 

Action of vasopressin and other hormones 84 

Vasopressin 84 

Aldosterone and renin-angiotensin system.. 85 

Cardioglobulin 86 



CONTENTS 



IX 



Page 

Laboratory of metabolism 86 

Pathway and inhibitors of cholesterol biosyn- 
thesis 87 

Normal pathway of cholesterol biosyn- 
thesis 87 

Desmosterol reductase 87 

Desmosterol as precursor of adrenal ste- 
roids and bile acids 88 

Sterol metabolism in skin and optic lens.. 88 

Atherogenicity of desmosterol - 88 

Metabolism of adipose tissue as influenced by 

hormones 88 

Factors controlling mobilization and utiliza- 
tion of FFA in vitro 89 

Relation between FFA utilization and meta- 
bolic rate 89 

Metabolic fate of fatty acids of different 

structure _ 90 

Serum lipoproteins and metabolism 91 

Metabolism of cholesterol esters 91 

Experimental nephrosis 91 

Protein structure 92 

Phospholipid biosynthesis in red blood cells 92 

Formation of chylomicrons from endogenous 

sources 92 

Action of parathyroid hormone 92 

Section on chemistry 93 

Gas phase chromatographic methodology. 93 

Alkaloid work 93 

Kallikrein-kallidinogen-kallidin system 93 

Informal collaborative research 93 

Laboratory of clinical biochemistry 94 

Am ine biogenesis and metabolism 94 

Collagen and hydroxyproline 95 

Proteins and peptides 95 

Amino acid uptake by animal tissues 96 

Biosynthesis of phospholipids and other lipids— 97 

Vitamin B 12 97 

Development of analytical procedures 98 

Section on biochemical genetics 98 

RNA and genetic code 98 

Cell-free assay for messenger RNA.. 98 

Messenger role of viral RNA 98 

Fate of messenger RNA 99 

Characteristics of genetic code 99 

Clinical endocrinology branch 99 

Adrenal function 99 

Calcium and phosphorus metabolism 101 

Experimental therapeutics branch 104 

Biochemistry of aromati c amines 104 

Chemical pharmacology and therapeutics 105 

Metabolism of hydroxyproline and collagen 106 

Miscellaneous 107 

Cardiology branch 107 

Adrenergic nervous system 107 

Biosynthesis and metabolism of norepine- 
phrine by the heart 107 

Subdivisions of norepinephrine pool 107 

Pharmacology of adrenergic blocking 

agents 108 



Page 
Cardiology branch — Continued 

Adrenergic nervous system — Continued 

Autonomic nervous system in heart failure, 

shock, anemia 109 

Action of sympathomimetic drugs 109 

Dynamics of ventricular contraction 109 

Cineradiographic measurements of ven- 
tricular dimensions 109 

Left ventricular function 110 

Digitalis 110 

Effects on nonfailing human heart 110 

Effectiveness of cardiac glycosides 111 

Cardiovascular physiologic studies 111 

Clinical cardiology 112 

Section on clinical biophysics 113 

Cardiovascular activities 113 

Pulmonary mechanics 115 

Clinic of surgery 115 

Gerontology branch 119 

Aging in the human 120 

Institutional and community residing sub- 
jects 120 

Physiological bases of behavior 121 

Age changes in psychological performance. 121 

Dietary intakes 121 

Renal physiology 121 

Endocrinology 122 

Carbohydrate metabolism 122 

Biology of aging 123 

Behavioral changes with age 123 

Effects of nutrition and parental age on 

longevity 124 

Effect of environment temperature on 

longevity 125 

Effect of radiation on longevity 125 

Aging in Cnidaria (Coelenterata) 125 

Age pigment 125 

Basic biology 126 

Cell division 126 

Oxidative phosphorylation 127 

Molecular structure: DNA 127 

Deoxyribonuclease 128 

Age changes in collagen 128 

National Institute of Arthritis and Meta- 
bolic Diseases 129 

Basic Research 129 

Introduction 129 

Laboratory of molecular biology 130 

Glutamic dehydrogenase 130 

Enzyme induction in mammals 130 

Enzyme induction in bacteria 130 

DNA-dependent RNA polymerase 130 

Histidine in salmonella 130 

Natural and synthetic polyribonucleotides 130 

Nucleic acids and components 130 

Hemoglobin 131 

Bacteriophage lysozymes 131 

Guanylic acid 131 

Synthetic polynucleotides 131 

Crystalline chymotrypsin. 131 



CONTENTS 



Page 

Laboratory of molecular biology — Continued 

Polynucleotide structure — melting point dis- 
persion 131 

Micrococcal nuclease 131 

Helix-coil transformations 131 

Histidine biosynthesis 132 

Laboratory of biochemical pharmacology 132 

Spermine and spermidine 132 

Sialic acids 132 

Sulfur-containing compounds 133 

Amino acids in bacteria 133 

Enzyme levels in mammalian systems 133 

Histidine, histamine, related imidazoles and 

amines 134 

Burns 134 

Mevalonic acid 134 

Leprosy 134 

Fatty changes in mice 135 

Laboratory of nutrition and endocrinology 135 

Vitamin E, antioxidants and selenium 135 

Protein deprivation and energy metabolism 135 

Lipid metabolism 135 

Guinea pig nutrition 136 

Diabetes and fat metabolism 136 

Protein hormones ; 137 

Folic acid 138 

Large-scale laboratory 138 

Germ-free program 138 

Laboratory of physical biology 139 

Physiology 139 

Molecular structure 139 

Protein structure, activity, synthesis 140 

Biological energy 140 

Laboratory of chemistry 141 

Rotatory dispersion of nucleosides 141 

Cz's-nucleosides 141 

Octuloses and nonuloses 142 

Anhydroheptuloses 142 

Rearrangements of cyclitols 142 

Glycosyl cyanides 142 

Benzomorphans 142 

Phenolic hydroxyl in a- and ^-benzomorphans. 143 

/3-benzomorphans, more potent series 143 

Codeinone series 143 

Analgesics 144 

Morphine tolerance 144 

Rapid test for addiction 144 

Synthetic antigens 144 

Chemistry of narcotine 144 

Quinozaline glucosides 144 

Acti ve center of ribonuclease 144 

Tertiary structure of proteins 145 

Configurational tautomerism of cyclopeptides_ 145 
Rapid configurational analysis of peptide 

hydrolysates 146 

Chemistry of Gramicidin A 146 

4-hydroxyproline and 7-hydroyornithine 146 

Cis- and <rans-hydroxyproline 146 

Congener of actinomycin 146 



Page 
Laboratory of chemistry — Continued 

Dehydrobufotenine in parotid gland of Bufo 

marinus 146 

Venom of arrow poison frogs 147 

Dopamine-/3-hydroxylase 147 

Catecholamaine metabolites 147 

Chemistry of adrenaline 147 

N-methylation of a purine 147 

Tocopherol and ubiquinones 147 

Dienone-phenol tautomerism 147 

Nucleic acids 148 

Sea cucumber poison 148 

Sapogenins 148 

Carcinogenicity and structure 148 

Chemistry of cortisone 148 

Steroids of insects 148 

Steroid biosynthesis in plants 149 

Microbial hydroxylation of steroid alkaloids. _ 149 

Steroids in cell membranes 149 

Steviol in plants 149 

Florigen, the flowering hormone 149 

Qualitative analysis of steroids 149 

Automatic steroid analyzer 149 

Infrared fine structure of steroids 150 

Microanalytical services 150 

Laboratory of biochemistry and metabolism 150 

Carbohydrate metabolism 150 

Carbohydrate polymers 150 

Small carbohydrate molecules and carbo- 
hydrate-containing coenzymes 150 

Other studies on biosynthesis 151 

Thiamine 151 

Fatty acid synthesis 151 

Regulatory mechanisms and hormones 152 

Nucleic acids and other polynucleotides 152 

Biochemical studies of lysogeny 153 

Enzymatic utilization of model compounds 153 

Laboratory of experimental pathology 153 

Anatomical pathology 153 

Altitude studies 153 

Bacterial endocarditis 154 

Cytogenetic studies 154 

Carcinogenicity of Cycas circinalis L 155 

Enzyme histochemistry 155 

Eosinophilic meningoencephalitis 156 

Experimental obesity 156 

Hematology 156 

Hemoglobin 156 

Histochemistry of mucopolysaccharides 157 

Hypersensitivity 158 

Immunochemical studies i 158 

Glyceraldehyde-3-phosphate dehydroge- 
nase 158 

Prolactin 158 

Juxtaglomerular apparatus 158 

Melanoma 158 

Cardiac lesions in carcinoid syndrome 159 

Monoamine oxidase inhibitors 159 

Pathology of rheumatic disease 159 



CONTENTS 



XI 



Page 
Laboratory of experimental pathology— Continued 

Fine structure 160 

Factor 3 — selenium 160 

Respiratory decline, sulfhydryl groups, toco- 
pherol 161 

Glucose tolerance, chromium (III), other fac- 
tors 162 

Office of mathematical research 162 

Clinical investigations 164 

Arthritis and rheumatism branch 164 

Sjogren's syndrome with malignant lym- 
phomas 164 

Organ distribution of lymphocytes 165 

Antigenic composition of glutamic dehydroge- 
nase 165 

Steroid hormone and enzymes 166 

Demography of rheumatic diseases. Genetic 

and environmental influences 167 

Population survey of Blackfeet Tribe 167 

Marianas Islands survey 168 

National Health Examination survey 168 

Antirheumatic drugs in rheumatoid arthritis, 

psoriatic arthritis and lupus nephritis 168 

Hydroxychloroquine in rheumatoid ar- 
thritis 168 

Corticosteroid therapy in lupus nephritis. _ 1 69 
Enzymatic defect in histidinemia, metabolism 
of aromatic amino acid and homogentisic 

acid in phenylketonuria, and ochronosis 170 

Phenylketonuria 170 

Histidinemia 170 

Experimental ochronosis and ochronotic 

arthritis 170 

Folic acid and tyrosine metabolism 170 

Tyrosine transaminase 171 

Gout 171 

Action of colchicine 171 

Uricolysis by human leukocytes 172 

Gastroenterology unit.. 172 

Whipple's disease 172 

Metabolism of D-xylose 173 

Amino acid transport in the small intestine 173 

Glucose metabolism by small-intestine mucosa. 173 

Clinical endocrinology branch 174 

Biochemistry of thyroid 174 

Iodide transport 174 

Thyroxine and iodotyrosine synthesis 175 

Iodoproteins 175 

Thyroxine transport in blood 176 

Chromatographic separation of iodoamino 

acids 176 

Action of thyroxine on isolated systems 177 

Congenital goiter 177 

Physical chemistry of proteins 177 

Carbohydrate metabolism 178 

Glucose 178 

Glycogen storage disease 178 

Mechanism of action of insulin on liver 178 

Galactose and galactosemia 179 

Amino acid transport 179 



Page 

Clinical hematology branch 180 

Immunologic studies 180 

Leukocyte isoantigen systems 180 

Significance of maternal antibodies against 

isoantigens on leukocytes and platelets. _ 180 
Clinical significance of isoantibodies against 

leukocytes 181 

Blocking reaction used to determine ob- 
scure isoantibodies 181 

Neonatal thrombocytopenic purpura 181 

Effects of isoantibodies on leukemic cells. _ 182 

Coagulation studies 182 

Measuring minimum in vivo concentra- 
tions of Factor VIII 182 

Standardization of methods of measuring 

Factor VIII 182 

Unusual form of painful purpura 183 

Acquired hemophilia due to abnormality 

in gammaglobulin 183 

Changes in fibrinogen levels in familial 

Mediterranean fever 183 

Pediatric metabolism branch 184 

Cystic fibrosis of pancreas 184 

Macromolecules in normal controls and patients 

with cystic fibrosis of pancreas 184 

Glycoproteins in sweat of cystic fibrosis patients 

and normal controls 184 

Biosynthesis of mucoproteins 184 

Glycogen storage disease 185 

Intestinal malabsorption in children 185 

Metabolic diseases branch 185 

Mineral metabolism studies 185 

Nutritional factors — dietary calcium in- 
take in osteoporosis 185 

Nutritional and hormonal influence on 

bone metabolism 186 

Energy metabolism studies 188 

Physiological studies of obesity 188 

Pharmacologic agents effect on fat metab- 
olism and metabolic rate 189 

Physiologic studies of temperature regu- 
lation 189 

Studies of exercise or work physiology 191 

National Institute of Allergy and Infectious 

Diseases 193 

Intramural research program 193 

Rocky Mountain laboratory 195 

Rocky Mountain spotted fever 195 

Tick paralysis 195 

Panama mite studies 195 

Colorado tick fever 196 

Phlyctenulosis 196 

The encephalitides 196 

Q fever 196 

Selected zoonoses of regional importance 197 

Viruses and chronic disease 197 

Allergy, its mechanisms and role in disease 197 

Fine structure of micro-organisms 197 

Structure and biologic activity of endotoxins. 198 

Biologic properties of Bordetella pertussis 198 



XII 



CONTENTS 



Page 
Rocky Mountain laboratory — Continued 

Biologic behavior of microbial proteins and 

nucleic acids 198 

Laboratory of germf ree animal research 199 

Penicillin not toxic for germf ree guinea pigs.- 199 

Death of germfree guinea pigs 199 

Pulmonary tumors 199 

Death in conventional and germfree mice 200 

Tissue reactions to mouse nematode 200 

Thyroiditis 200 

Laboratory of biology of viruses 200 

Immunologic studies 200 

Genetics 201 

Cell metabolism 201 

Biochemistry of viral replication 201 

Biophysical studies 202 

Laboratory of tropical virology 202 

Vesicular stomatitis virus 202 

Sandfly fever in the New World 202 

Hemorrhagic fever in Bolivia 202 

Encephalitis viruses in Panama 202 

Bunyamwera group of viruses 202 

Pathogenic agents from Panamanian acarina.. 203 

VEE vaccination 203 

Serologic reagents from ascitic fluid 203 

Leptospirosis in Panama 203 

Bats and histoplasmosis 203 

Laboratory of immunology 203 

Cross reactions in human malaria disclosed by 

fluorescent antibody 203 

Allergy reagins identified as beta 2 A-globulins_ 204 
Physiological basis for susceptibility to anaphy- 
lactic shock 204 

Delayed type hypersensitivity and allergic 

thyroiditis 204 

Implications of allotypy for experimental bi- 
ology 204 

Inhibition of protein synthesis in rabbits 204 

Cellular production of gamma globulin allo- 
types shown by fluorescent antibody 205 

Allot3'pes shown to be genetic markers 205 

Genetic control of allotypes related to chemical 

structure 205 

New mouse allotypes discovered by ascitic 

fluid technique 205 

Allotypes found in man 206 

New methodology for characterizing enzymes. 206 
Action of immunosuppressive drugs on normal 

serum globulins 206 

Delayed allergy with in vitro techniques 206 

Laboratory of clinical investigations 207 

Purine antimetabolites in nonneoplastic dis- 
eases 207 

Toxicity of 6-thioguanine 207 

Action of 6-thioguanine 207 

Respiratory viral agents in normal volunteers. 208 

Anti-viral therapy 208 

Malaria 208 

Biochemical assay for penicillin 208 



Page 

Laboratory of clinical investigations — Continued 

Fungus disease 209 

Plasma cell tumors and antibody production.. 209 

Laboratory of parasitic diseases 209 

Angiostrongylus cantonensis 209 

Experimental schistosomiasis 209 

Snail hosts of schistosomes 209 

Antimony compounds and diet in treatment. . 210 

Axenic cultivation of amoebae 210 

Biochemistry of parasites 210 

Toxoplasmosis 210 

Laboratory of bacterial diseases 211 

Pleuropneumonia-like organisms 211 

Intracellular parasitism 211 

Laboratory of parasite chemotherapy 211 

Malaria — human 211 

Malaria — simian 211 

Biochemical studies 212 

Immunological studies 212 

Far East research project studies in Malaya. . 212 

Schistosomiasis 213 

Virus-mosquito larvae associations 213 

Intestinal parasites 213 

Laboratory of infectious diseases 213 

Adenovirus types 12 and 18 as cancer viruses. _ 213 

Papilloma viruses 213 

Natural history of polyoma virus 213 

Respiratory virus and vaccine studies 214 

Complexity of the rhinoviruses ... 214 

Mycoplasma agents in respiratory disease 214 

Virus infections and human cancer 214 

Cancer viruses in vaccines and foodstuffs 214 

Pacific research laboratory, Hawaii 215 

Rubella virus isolation in tissue cultures 215 

Enterovirus studies 215 

Antibacterial effect of sea water 215 

Studies on hydrogenomonas 215 

Siderophilin 215 

Bacteriophage treatment for urinary infections. 215 

Detoxification 216 

Cell wall formation 216 

Environmental sources of infection in mycoses. 216 

Chemotherapy of mycoses 216 

Physiology of Coccidioides immitis 216 

Immunity in the mycoses 217 

Cryptococcus antigen 217 

National Institute op Mental Health 219 

Intramural Research 219 

Introduction 219 

Clinical investigations 220 

Laboratory of clinical science , 223 

Basic biological research 223 

Catecholamines 223 

Biological transmethylation 224 

Action of thyroxine 224 

Autosensitization phenomena in central 

nervous system 225 

Biochemical aspects of membrane func- 
tion 225 



CONTENTS 



XIII 



Page 
Laboratory of clinical science — Continued 
Basic biological research — Continued 

Individual cortical neurons in sleep and 

waking 225 

Clinical biology 226 

Production, metabolism, excretion of 

catecholamines in man 226 

Changes in blood levels of free fatty acids 

(FFA) 226 

Clinical physiology 227 

Psychiatry 227 

Schizophrenia 227 

Acute psychotic episode 228 

Aging 228 

Mental retardation 228 

Laboratory of psychology 229 

Office of chief 229 

Section on early development 231 

Section on perception and learning 233 

Section on personality 234 

Creativity 235 

Group creativity variables 235 

Effects of psychotherapy on produc- 
tivity 235 

Motivational factors 235 

Formal characteristics of speech 237 

Section on neuropsychology 237 

Perceptual functions of posterior associa- 
tion cortex 237 

Vision 237 

Olfaction 238 

Audition 238 

Somesthesis 238 

Problem-solving functions of frontal asso- 
ciation cortex 239 

Emotional-motivational functions of the 
limbic system. Forebrain alimentary 

system 239 

Interaction study 239 

Intensity study 240 

Alerting functions of reticular activating 

system 240 

Section on aging 240 

Unit on psychophysiology 241 

Unit on higher cognitive processes 241 

Animal studies 242 

Laboratory of socio-environmental studies 244 

The family 244 

Parent-child relationships; personality de- 
velopment of child 244 

The family in Japan 246 

Social class and parent-child relationships. 247 
Social class and parent-child relationships 

in Italy 247 

The mental hospital and psychiatric patients. 248 

Social structure of the mental hospital 248 

Behavior and characteristics of patients. . 248 

Job, occupation, and career 249 



Page 
Laboratory of socio-environmental studies — Con. 

Relevant social variables 250 

Self-esteem among adolescents 250 

Social psychology of aging 250 

Addiction research center 251 

Addictive properties of new analgesics 253 

Intoxication with drugs other than analgesics 

and barbiturates 254 

Intoxication with alcohol, barbiturates and 

related drugs 255 

Biochemistry of addiction 256 

Morphine and the catecholamine level of 

rat organs 256 

Addiction 257 

Abstinence 257 

Future experiments 257 

Excretion of catecholamines 257 

Morphine-induced hyperglycemia in dogs. 257 
Neurophysiology and neuropharmacology of 
chronic intoxication with barbiturates and 

related drugs 257 

Elevation of electrical seizure thresholds. 257 
Decerebellation and barbiturate with- 
drawal convulsions 258 

Nondepressant chemical and barbiturate 

abstinence convulsions 258 

Psychological studies of addiction 258 

Mode of action of central nervous system 

depressants 259 

Conditioning factors in addiction and habitua- 
tion 261 

Morphine intoxication, morphine absti- 
nence syndrome 262 

Oral consumption of etonitazene solution. 262 

Opoid (etonitazene) drinking behavior 263 

Psychophysical studies 264 

Social science 265 

Laboratory of cellular pharmacology 266 

Laboratory of neurobiology 268 

Scientific program 268 

Physical analysis of excitability 268 

Mathematical analysis of visual percep- 
tion 269 

Sensory and nonsensory corticipetal 

pathways 270 

Longitudinal organization of sensorimotor 

coordination 270 

Somatic and autonomic convergence on 

vagal outflow 272 

Electrical activity of hypothalamic feeding 

center 272 

Action of tympanic muscles 272 

Physiological effects of prolonged sensory 

stimulation 273 

Administration of the laboratory 275 

Laboratory of neurochemistry 275 

Laboratory of neurophysiology 277 



XIV 



CONTENTS 



Page 

Clinical neuropharmacology research center 279 

Clinical program developments 279 

Clinical psychopathology 280 

Clinical psychopharmacology 280 

Differential values of somatic therapies 281 

Psychotherapy 281 

Psychosocial modes of treatment 281 

Family studies 282 

Catamnestic followup studies 282 

Concept and functions of model psychi 

atric clinic 282 

Information processing in man 283 

Central nervous system processing of com- 
plex auditory information 283 

Motor control system in man 283 

Sensory feedback in diseases characterized 

by abnormal movements 284 

Catecholamine metabolism 285 

Release of epinephrine from adrenomedul- 

lary particles in vitro 285 

Catecholamine excretion patterns in space 

flight 285 

Urinary excretion of catecholamines and 

metabolites 285 

Catecholamine metabolites in rat urine 285 

Psychoactive tryptamine derivatives 285 

Nicotinamide adenine dinucleotides (NAD) in 

rat liver 287 

Intermediate metabolism of chlorpromazine 

and related compounds 288 

Metabolism of chlorpromazine in man 288 

Pharmacological and behavioral effect of 
metabolites of chlorpromazine and pro- 
mazine 288 

Forrest and Forrest "FPN" test for uri- 
nary phenothiazines 288 

Reversible isolation of brain areas by short- 
term cooling 289 

Microelectrode and micropipette studies on 

discharge patterns 289 

Miscellaneous animal behavior studies 290 

Adult psychiatry branch 291 

Schizophrenics and their families 291 

Adolescent development 293 

Experiment in training mental health coun- 
selors 295 

Stress and anxiety 296 

Sleep and dreaming 297 

Parents of leukemic children 298 

Depression 299 

Neurochemical factors in behavior 300 

Child research branch 300 

Program summary 300 

Behavior continuities from infancy to age 

three 301 

Initial marital patterns of adaptation 303 

Initial parent-infant patterns of adapta- 
tion 304 

Table: timetable for the longitudinal program, 305 



Page 
National Institute of Neurological Diseases 

and Blindness 307 

Report of the clinical director 307 

Medical neurology branch 307 

Introduction 307 

Neuromuscular disease 307 

Pharmacologic investigations 310 

Neuroradiologic studies 311 

Bony structures 312 

Structures delineated by intravascular con- 
trast material 312 

Soft tissues 312 

Radioisotopes 312 

Surgical neurology branch 313 

Epilepsy 313 

Involuntary movements 314 

Developmental defects 314 

Cerebral edema 314 

Cerebral trauma 315 

Low temperatures 315 

Anesthesia 315 

Surgical lesions 316 

Radiant energy 316 

Technical developments 316 

Language and memory 317 

Ophthalmology branch 318 

Electroencephalography and clinical neurophysi- 
ology branch 323 

Diagnostic service 323 

Research activity 324 

Other activities 325 

Laboratory of neuroanatomical sciences 325 

Laboratory of neuropathology 329 

Laboratory of neurophysiology, intramural re- 
search 330 

General neurophysiology 330 

Section on spinal cord 330 

Laboratory of biophysics, intramural research 331 

Laboratory of neurochemistry 332 

Laboratory of molecular biology, intramural re- 
search 334 

Major results of research 335 

Mutations in transforming DN A 335 

Temperature and pH dependence of DNA 

melting and rate of uncoiling 336 

Head protein of T-bacteriophages 336 

Enzyme synthesis 336 

National Institute of Dental Research 339 

Introduction 339 

Laboratory of histology and pathology 342 

Biophysical research 343 

Crystal chemistry 344 

Histochemistry and experimental pathology 345 

Laboratory of microbiology 345 

Periodontal disease 345 

Dental caries 346 

Microbial taxonomy 347 

Microbial physiology 347 

Immunology 347 

Virology 348 



CONTENTS 



XV 



Page 

Laboratory of biochemistry 349 

Protein chemistry 349 

Lathyrism 349 

Collagen, molecular weight, structure and 

function 349 

Calcification 350 

Dental caries — relation to phosphates 350 

Congenital malformation and teratogenic 

agents 350 

Saliva and salivary glands 351 

Enzyme chemistry 351 

Control of metabolic processes 352 

Epidemiology and biometry branch 352 

Nutrition surveys 353 

Fluorine and dental caries 354 

Other studies 354 

Clinical investigations branch 355 

Oral surgery and oral medicine 355 

Dental caries 355 

Human dental pulp 355 

Periodontal disease investigations 356 

Anesthesia studies 356 

Soft tissue lesions 356 

Human genetics 357 

Oral pharyngeal development 359 

Activities 360 

Activities of dental services section 361 

With National Cancer Institute 361 

With National Heart Institute 361 

With National Institute of Arthritis and 

Metabolic Diseases 361 



Page 

Division of Biologics Standards 363 

Introduction 363 

Laboratory of control activities 365 

Laboratory of bacterial products 366 

Allergenic products 366 

Bacterial toxins 367 

Bacterial vaccines 367 

Nonresearch activities 367 

Laboratory of viral immunology 367 

Live polio virus vaccine 367 

Adenovirus vaccine 368 

Immune serum globulin 368 

Measles vaccines 368 

SV-40 368 

Laboratory of virology and rickettsiology 369 

Measles 369 

Avian leukosis virus complex and the RIF test. 370 

Hepatitis 370 

Continuous cell culture lines 371 

Rickettsial vaccines 371 

Influenza 372 

Arboviruses 372 

Antimicrobial substances 372 

Laboratory of biophysics and biochemistry 372 

Laboratory of blood and blood products 373 

Research activities 373 

Other research interests 373 

Offsite Research 373 



NATIONAL CANCER INSTITUTE 



INTRODUCTION 

The major studies of the clinical branches of 
the National Cancer Institute are an evaluation 
of chemohterapeutic methods for the treatment 
of neoplastic disease; the use of radical surgery 
in carcinoma of the uterus and head and neck; 
a study of the natural history of neoplastic dis- 
eases, particularly as modified by various thera- 
peutic procedures; studies of the biochemical and 
physiological changes produced in the host by 
a tumor, and studies involving the use of experi- 
mental animals as a supplement and complement 
to the studies carried out in man. 

SPECIFIC HIGHLIGHTS 

Specific highlights in the last year are an in- 
crease in the remission rate in choriocarcinoma to 
75% ; the introduction of methyl glyoxal-bis- 
guanylhydrazone (the first drug to be active in 
a large percentage of patients with acute myel- 
ogenous leukemia) ; and the introduction of 
vincristine (a new agent that is active in the 
treatment of acute lymphatic leukemia). These 
are the only new agents to be introduced into the 
therapy of acute leukemia in the last ten years. 
A very active urological service has been de- 
veloped which provides urological consultation 
within the National Cancer Institute and through- 
out the other institutes. In addition, a stimulating 
and productive urology research program has been 
initiated. A monkey colony has been established, 
under contract, for pharmacological and carcino- 
genesis studies. 

NATURAL HISTORY OF THE DISEASE 

Acute Leukemia 

The principal complications of acute leukemia 
are infection and hemorrhage. The procurement 
of platelets and their transfusion in large quanti- 
ties represents a major research and therapeutic 
effort. It has been shown that platelets can be 

705-685—63 2 



procured, can be transfused in large quantities 
and that bleeding complications can be averted. 
Further studies in platelet storage are essential to 
make this technique more widely available in clini- 
cal medicine. The use of white cells derived from 
patients with chronic myelocytic leukemia has re- 
sulted in a very marked reduction in the mortality 
associated with Pseudomonas septicemia, thus 
averting another one of the principal causes of 
death in acute leukemia. 

Multiple Myeloma 

A review has been completed of the natural his- 
tory of this disease. Of particular interest was 
the inability to correlate the clinical findings with 
the type of myeloma protein produced. There was 
no difference in the median survival of patients 
with gamma myeloma, Beta 2A , or Bence-Jones 
protein producing myelomas. There were statis- 
tical differences in the level of serum protein, the 
extent of bone disease and clinical symptoms be- 
tween the gamma myeloma protein producing and 
the non-gamma myeloma protein producing pa- 
tient groups. Studies are being carried out on the 
effect of chemotherapy in experimental animals 
with a plasma cell tumor. Cytoxan and 5-Fluo- 
rouracil were shown to be effective in producing 
an improvement in survival and elimination of 
serum protein abnormality. Other agents pro- 
duced prolongation of survival time but only in- 
frequently was there an improvement in the serum 
protein abnormality. Urethane improved sur- 
vival time but was without effect on the serum pro- 
teins. Large doses of androgens have been used in 
multiple myeloma. In four of five patients there 
was an increase in the rate of formation of red 
cells. There also appears to be some increase in 
the white blood cell count. L-phenylalanine mus- 
tard was shown to be effective in approximately 
40% of 40 patients with multiple myeloma, with 
an increase in hemoglobin, improvement in the 
bone marrow and bone healing. Patients with 
multiple myeloma showed an increased suscepti- 
bility to infection due to impairment of antibody 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



production. There was no correlation between 
protein types and degree of immune deficiency. 
Significant advances were made in the ability to 
diagnose by immunochemical means the types of 
proteins produced and to identify the cells produc- 
ing these proteins. 

Head and Neck Cancer 

A review of 42 patients dying of epidermoid 
carcinoma of the head and neck region disclosed 
that 55% had distant metastases, almost entirely 
to the lung. This high incidence of metastases is 
primarily due to a prolongation of life by control 
of the primary lesion as a result of radical surgery. 
There appears to be a good correlation between 
epidermoid cancer of the mouth and pharynx with 
combined alcohol and tobacco consumption. 

Cervical Cancer 

Two hundred patients with a diagnosis of carci- 
noma of the crevix have been followed for at least 
one year after therapy. Fifty-two percent of these 
patients had received previous X-ray therapy or 
surgery ; 39 of these patients were treated by radi- 
cal hysterectomy ; 25 had anterior pelvic exentera- 
tions, and 71 had total pelvic exenterations. The 
survival rate of five years was 57% for both radi- 
cal hysterectomy and anterior pelvic exenteration 
groups, and only 16% for the total pelvic exentera- 
tion group. The overall survival rate was 37% 
at 5 years. 

Infection in Surgery 

Clinical surgery continues to be concerned with 
post-operative infections. The infection rate fol- 
lowing extensive cancer surgery decreased from 
approximately 30% in 1956 to a low of approxi- 
mately 10% in 1962, but the percentage of infec- 
tions that were staphylococcal increased from ap- 
proximately 35% in 1956 to approximately 70- 
80% in 1962. This decrease incidence was brought 
about by the introduction of both a planned pro- 
gram of antibiotic prophylaxis and increased 
awareness of the staff to this problem. 

Theory of Basal Cell Cancer 

A tentative definition of the biogenesis of basal 
cell carcinoma has been developed. These cells 



fail to keratinize. Because of this, the mecha- 
nisms by which the cell is enabled to move from 
the basal layer to the surface is lacking, causing the 
basal cells to accumulate as a tumor mass. This is 
an interesting concept requiring further investi- 
gation. 



THERAPY 

Evaluation of ureteroileostomy in extensive 
pelvic surgery, studies of circulating tumor cells 
and wound seeding continue to be major efforts. 

The effects of a single dose of radiation therapy 
of the order of 2,000-2,500 Roentgens for treat- 
ment of patients with primary cancer of the 
mouth, pharynx or larynx have been investigated. 
There was immediate disappearance of the pri- 
mary cancer in 18 of 20 patients. At 2,000 R, 
mild local reactions were observed. At 2,500 R, 
there were severe local reactions in all of the 11 
patients treated. There has been one recurrence of 
the primary lesion at primary radiation site. This 
study indicates that 2,500 R, in a single dose, ex- 
ceeds tolerance . The tissue reactions at 2,000 R 
were those which were expected. 

Chemotherapy is a major clinical endeavor of 
the National Cancer Institute. The principal 
findings in the last year : (1) demonstration that 
methylglyoxal-bis-guanylhydrazone is effective in 
the treatment of acute myeloblastic leukemia; (2) 
the findings that vincristine was effective in the 
treatment of acute lymphoblastic leukemia; and 
(3) the demonstration that a combination of meth- 
otrexate and Actinomycin D can raise the remis- 
sion rate to 75% in choriocarcinoma. 

The primary treatment of non-metastasizing 
trophoblastic disease with chemotherapeutic meth- 
ods has resulted in complete remission in 13 of 15 
patients studied, thus preserving the uterus for 
further child bearing. In two patients complete 
remission was achieved by hysterectomy after 
chemotherapy. A combination of four agents 
(vincristine, folic acid antagonist, 6-mercapto- 
purine, prednisone) with the provocative initials 
VAMP, has produced early remissions in acute 
leukemia. Other new agents that have been tested 
are: hydroxyurea, nitrosourea, ethiochloanolone, 
azauridine, and terephthalanalides. 



NATIONAL CANCER INSTITUTE 



BIOCHEMICAL AND PHYSIOLOGICAL 
STUDIES 

Im in iinological 

A number of studies are immunologically ori- 
ented. These include determination of the chem- 
ical structure of the gamma globulins by means 
of enzymatic and chemical degradation, and a 
comparison of the three types of gamma globulin 
associated with myeloma and macroglobulinemia. 

Patients who were incurable by conventional 
modes of therapy but in good general condition 
have been immunized by intradermal and intra- 
muscular injections of their own frozen killed 
tumor in Freund's adjuvant. In two of the seven 
patients studied, there was an inflammatory re- 
sponse with degenerative changes in the tumor 
cells. The response has also been evaluated by 
means of skin tests, immunofluorescent studies and 
the use of various hemagglutination and cytotox- 
icity methods. Tumor from 10 patients with my- 
cosis fungoides has been injected in a horse and 
the serum harvested. This serum showed a reac- 
tion to mycosis fungoides tumor tissue by immu- 
nochemical tests. When this serum was given to 
3 patients intratumorally there was necrosis of 
tumor at the site of injection but no comparable 
reaction was observed when this material was in- 
jected into normal skin of the same patients. 

Steroid Chemistry 

Gas liquid chromatography has been used for 
the development of a rapid and reliable quantita- 
tion of the chief urinary 17-ketosteroids. It is 
anticipated that several problems concerning keto- 
steroid excretion can now be studied. Gas liquid 
chromatography has also been used to study the 
excretion of dehydroepiandrosterone. A fluoro- 
metric method for the determination of testoster- 
one in small quantities has been developed which 
makes it possible to measure testosterone secretion 
rates. A simplified method has been described for 
the clinical determination of urinary corticoids. 

Pyrimidine Metabolism 

By measurement of the rate of excretion of 
pseudouridine and the conversion of Carbon 14 
labeled orotic acid to pseudouridine, an estimate of 
total pyrimidine metabolism can be made. This 
method is comparable in its application to the 
measurement of urinary uric acid excretion to 



quantitate purine metabolism. These studies have 
shown a disproportionately increased pyrim- 
idine synthesis in chronic lymphatic leukemia. 
6-Azauridine produces both a large increment in 
total pyrimidine synthesis and an apparent in- 
crease in the synthesis of decarboxylation products 
from orotic acid. 

Pharmacology 

To support the chemotherapeutic efforts, a num- 
ber of pharmacological studies have been carried 
out. These consist of both toxicological and bio- 
chemical pharmacology. Several of the newer 
agents have been prepared in radioactive form and 
their disposition in experimental animals and man, 
and in some cases metabolic fate in terms of con- 
version to other chemical compounds studied. 

Normal Skin 

Psoriasis has been shown to be due to a ninefold 
accelerated proliferation of the epidermal cells, 
and is due mainly to an expanded population of 
germinative basal cells per unit area of surface 
epidermis, rather than due to an increased mitotic 
rate of the normal complement of germinative 
cells. In the normal and psoriatic epidermis mi- 
tosis is confined to cell layers in immediate prox- 
imity to supporting connective tissue. In the hair 
matrix and in basal cell tumors, mitosis occurs in 
cells more or less randomly distributed in the 
epithelial population and in areas comparatively 
distant to supporting connective tissue. 

Lymphangiography 

The injection of a contrast medium into the lym- 
phatics of either the foot or the hand and serial 
X-ray examination of regional areas has provided 
a technique for visualization of lymphatic chan- 
nels and lymph nodes in over 200 patients. A high 
degree of accuracy in the prediction of lymph node 
involvement in cancer and in the diagnosis of en- 
larged nodes in patients with lymphoma has been 
achieved. This technique has proven to be of 
sufficient value that we are recommending to the 
Diagnostic Radiology Department that it be 
adopted as a procedure to be available routinely 
in their department. 

White Cells and Platelets 

White blood cells and platelets are being used 
in large quantities. Studies of methods of pro- 



4 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



curement and storage are necessary. A centrifuge 
is being developed to separate white cells and 
platelets from red cells. A working model has 
been built and a considerable purification has been 
achieved. Additional working models are to be 
constructed and it is hoped that a centrifuge can 
be built that will provide for the efficient inline 
extraction of white cells and platelets from whole 
blood. 

Carcinogenesis 

Carcinogenesis has been studied in new born 
rodents and monkeys. 

METABOLISM SERVICE 

Amino Acid Transport 

Studies of amino acid transport in the kidney 
serve as a model for investigating the problem of 
cell membrane transfer or transport. The kidney 
is a convenient tissue to use since transport is a 
prominent process occurring in this organ. 

To develop an animal model system for the 
study of aminoaciduria, the ability of rat renal 
cortex slices to concentrate a variety of amino 
acids has been investigated. Steady state uptake 
curves for glycine, L-glycine, and alpha amino- 
isobutyric butyric acid have been fitted to a three 
compartment model system by means of a digital 
computer. These three compartments are me- 
dium, extracellular space, and intracellular space. 
Influx and efflux rate constants between these 
three compartments have been calculated and esti- 
mates of the rate of protein synthesis and conver- 
sion to carbon dioxide have been made. From 
these studies it was concluded that the rate of in- 
corporation of those amino acids studied into pro- 
tein does not depend upon equilibration with the 
intracellular amino acid pool. Growth hormone 
did not affect amino acid uptake or protein syn- 
thesis. No difference was observed in kidney tis- 
sues derived from normal and hypothesectomized 
rats. 

Maleic acid, a compound which has been noted 
to produce aminoaciduria, glycosuria and phos- 
phaturia in intact rats has been studied by in 
vitro techniques. Results of these in vitro studies 
corroborate in vivo findings. This suggests that 
maleic acid interferes with intracellular energy 
yielding processes required by active transport 
systems, rather than by competing with amino 



acids for membrane transfer. The effect of maleic 
acid could be prevented by the addition of sulf- 
hydryl groups. Although sulfhydryl groups had 
this effect, evidence for maleic acid exerting its 
effect by sulfhydryl binding could not be ob- 
tained and to the contrary — the maleic acid effect 
could be reversed by transfer to media not con- 
taining the acid. With labelled maleic acid, spe- 
cificity appeared to depend upon its failure to 
penetrate the cellular compartment in either 
muscle or intestine. 

A patient with elevated blood histidine and his- 
tidiunria level failed to excrete increased quan- 
tities of any other amino acid in the urine. The 
other members of the family who were studied had 
completely normal urinary amino acid excretion. 
These studies indicate that histidine failed to com- 
pete with other amino acids for tubular reab- 
sorption. 

Gamma Globulin Structure 

Most of the known functions of the immune 
system are effected by gamma globulin molecules. 
The structure of gamma globulin molecules is be- 
ing investigated in an effort to relate molecular 
structure to molecular function and to the genetic 
and biochemical properties of plasma cells. The 
gamma globulins, however, are unique among pro- 
tein systems in being very heterogeneous. There- 
fore, careful separation of the major normal 
gamma globulins subgroups,, study of the less het- 
erogeneous myeloma proteins and Bence-Jones 
proteins, and dissection of purified proteins into 
the substituent polypeptide chains was under- 
taken. Because of the complexity of the gamma 
globulins, terms such as 6.6Sy, /3 2 A and y x -micro- 
globulins are used to describe different classes of 
globulins and S and F pieces and "L" and "H" 
chains to describe subunits. 

Subunits of gamma globulin molecules were ob- 
tained by enzymatic fragmentation (S and F 
pieces) and by chemical reduction and alkylation 
("L" and "H" chains). "L" chains represent ap- 
proximately 25% of the gamma globulin mole- 
cule. Polypeptide chains of the "L" type were 
identified in all four classes of gamma globulins, 
i.e., in the 6.6S gamma globulins, /? 2 A-globulins, 
and yi-macroglobulins of serum and in the gamma - 
microglobulins of normal urine. In addition to 
normal gamma globulins, all of 140 myeloma pro- 
teins, macroglobulins and Bence-Jones proteins 



NATIONAL CANCER INSTITUTE 



formed in malignant plasma cells were found to 
have typical "L" polypeptide chains. Whether a 
protein was a 6.6S gamma globulin, a /3 2A -globulin 
or a y-macroglobulin was determined by the prop- 
erties of the H chains, represented in the F piece. 

The Bence-Jones proteins were shown to be 
antigenically and electrophoretically identical to 
"L" polypeptide chains obtained from myeloma 
proteins or macroglobulins of the same patient. 
Other subunits of myeloma protein, represented 
in the H chains and F pieces, however, were un- 
related to Bence-Jones proteins, and it appears 
reasonable to regard Bence-Jones proteins as ex- 
cessive quantities of "L" type polypeptide chains. 

Two types of "L" chains were identified in all 
classes of normal gamma globulins. The Type I 
and II "L" chains differ markedly in antigenic 
determinants and indicate differences in structural 
configuration of these two types of polypeptide 
chains. Approximately 65% of gamma globulins 
have Type I "L" chains, and 35% have Type II 
"L" chains. Bence-Jones proteins are either Type 
I or II polypeptide chains and, similarly, myeloma 
proteins and macroglobulinemic macroglobulins 
have either Type I or II, but not both types of 
"L" chains. Type I and II molecules appear to 
be synthesized in separate cell clones. 

The observations on gamma globulin structure, 
especially on Bence-Jones proteins and myeloma 
proteins, indicate several characteristic features 
of plasma cells. Plasma cells normally synthesize 
polypeptide chains in two forms ("L" chains and 
"H" chains). In man, two types of "L" chains 
and three types of "H" chains have been identified. 
Clones of malignant plasma cells synthesize only 
one of the two types of "L" chains and one (or 
none) of the three types of "H" chains. No malig- 
nant plasma cell clone forms all types of polypep- 
tide chains. It seems likely that normal plasma 
cell clones are similarly limited, and that the nor- 
mal plasma cell population is heterogeneous in re- 
spect to its capacity to synthesize gamma globulin 
molecules. 

Gamma Globulin Metabolism 

A combination of factors made possible a num- 
ber of studies of gamma globulin metabolism. 
These were (1) the capacity to prepare purified 
gamma globulin fractions (i.e., normal 6.6S 
gamma globulins, normal 18S gammai-macro- 
globulins and Bence-Jones proteins), (2) the ca- 



pability of measuring the serum levels of indi- 
vidual groups of gamma globulins, (3) the capac- 
ity to label purified proteins with I 131 without 
damage, and (4) availability of patients with a 
wide variety of changes in the gamma globulin 
components and of inbred strains of mice with 
protein producing plasma cell tumors. 

The metabolism of 6.6S gamma globulins (mol. 
wt. = 160,000) was established in subjects with 
normal gamma globulin levels. Forty-four per- 
cent of the total body 6.6S gamma globulin was 
intravascular and the total body gamma globulin 
content was approximately 1.22 grams per kilo- 
gram of body weight. The mean half time of 
gamma globulin survival (Ti/o) was 23 days and 
the fractional degradation rate was 3.0 percent of 
the body pool per day. The turnover rate for 6.6S 
gamma globulin was 0.036 grains per kilogram of 
body weight per day, i.e., approximately 1/7 that 
of albumin. 

In patients with multiple myeloma and large 
amounts of gamma-myeloma proteins the rate of 
gamma globulin catabolism was increased (with 
reduction in Ty 2 to 11 to 17 days) in 7 of 11 pa- 
tients, indicating that increased catabolism con- 
tributed to the hypogammaglobulinemia of these 
patients. In six patients with macroglobulinemia 
and four patients with Bence-Jones proteinuria, 
serum /3 £A -myeloma protein or no anomalous pro- 
tein, the serum and total body gamma globulin 
levels typically were reduced. The survival of 
gamma globulin was normal or prolonged (mean 
T%— 30.5 days) and the calculated synthetic rate 
was reduced (mean value =0.024 g/kg/day) indi- 
cating that impaired gamma globulin formation 
was the major cause of hypogammaglobulinemia 
in these patients. 

Two characteristic features of gamma globu- 
lin metabolism were defined by studies in mice and 
in man. Gamma globulin catabolic rate was 
shown to depend on the serum gamma globulin 
level. The fractional rate of gamma globulin 
catabolism decreased as gamma globulin levels 
fell, and increased as gamma globulin levels in- 
creased. A maximum rate of gamma globulin 
catabolism was approached as gamma globulin 
levels rose above 3 grams percent in both mouse 
and man, indicating a limit to the flexibility of 
gamma globulin catabolic processes. The factors 
determining gamma globulin catabolism were 
shown to be highly selective, i.e., specific for 6.6S 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



gamma globulin and not responsive to changes in 
the serum levels of the closely related proteins 
formed in plasma cells (gammax-macroglobulins 
or /? 2A -globulins) or of serum albumin. These 
observations indicate that a specific site on the 
gamma globulin molecule is responsible for re- 
moval of 6.6S gamma globulins. They also indi- 
cate that a specific homeostatic mechanism deter- 
mines gumma globulin catabolism and contributes 
to the maintenance of normal serum gamma globu- 
lin levels. 

Albumin Metabolism 

There is little definitive knowledge of the fac- 
tors that control the rate of catabolism of the serum 
proteins. Conflicting evidence suggests that the 
gastrointestinal tract, the liver, or even the kidney 
and reticuloendothelial system play the major 
roles in the catabolism of albumin. An increase in 
metabolic rate, fever, and cortisone administration 
accelerates the catabolism of albumin while a diet 
extremely deficient in nitrogen prolongs albumin 
survival. 

The distribution and metabolism of the serum 
proteins depends on both host factors and the na- 
ture of the protein. In an effort to evaluate the 
importance of host factors, the metabolism of a 
single preparation of I 131 labeled mouse albumin 
was studied in mice, rabbits, dogs and sheep. The 
mean survival T% of mouse albumin was IV2 days 
in mice, 6 days in rabbits, 10 days in dogs, and 
15 days in sheep, equivalent to the survival of 
autologous albumin in these species. This suggests 
that host factors play the major role of albumin 
survival in these species. 

The catabolic rate of a given protein can be 
modified without altering the survival of the re- 
maining proteins, suggesting that the individual 
proteins have unique catabolic pathways. Spe- 
cifically, it was shown that the survival of normal 
6.6S gamma globulin was 22-26 days Ty 2 in con- 
trol subjects and 30-70 days in patients with 
agammaglobulinemia, while the survival of I 131 
albumin was 17-20 days in both groups. A com- 
parable situation was seen with serum albumin. 
In two analbuminemic patients, there was a mark- 
edly prolonged survival of I 131 albumin but a 
normal survival of I 131 gamma globulin. 

In addition to the specific routes of catabolism 
for proteins nonspecific factors such as loss into 
the gastrointestinal tract appear to be a major 



factor in the normal catabolism of the protein, as 
well as the pathogenesis of the hypoalbuminemia 
seen in association with a variety of disorders. 
Intravenously administered Chromium 51 labeled 
albumin has been developed as a technique for the 
demonstration of gastrointestinal protein loss. It 
was shown to be a simple technique of value in the 
quantitation of protein loss, detection of its site 
and the early determination of therapeutic effec- 
tiveness of agents for the treatment of gastroin- 
testinal disorders. The major disadvantage of the 
technique lies in the short intravascular survival 
of the Chromium 51 labeled albumin. Of advantage 
over I 131 PVP are the nonabsorbability of the 
label, the ability to label a variety of serum pro- 
teins, and the greater ease of differentiation of 
normals from patients with excessive gastrointes- 
tinal protein loss. The use of I 131 albumin in con- 
junction with oral amberlite IRA 400 resin has 
been shown to be inadequate since the basic as- 
sumption of this technique that iodine be lost into 
the gastrointestinal tract only bound to albumin is 
not met. When NA I 131 was given intravenously 
and the resin and Lugol's solution by mouth, 40-50 
percent of the injected dose appeared in the sub- 
sequent stool collections. Turnover studies using 
I 131 labeled 6.6S and gamma macroglobulins indi- 
cate that these proteins are lost into the gastro- 
intestinal tract in those patients with albumin loss 
into the gut. In the case of 6.6S gamma globulins 
as with albumin, there is only a relatively small 
increase in the synthesis rate of the protein despite 
exceedingly low serum concentrations. Gastro- 
intestinal protein loss has been demonstrated, in 
cooperation with physicians of NHI and NIAMD, 
in patients with Whipple's disease, giant rugal 
hypertrophy, sprue, constrictive pericarditis, and 
congestive failure, agammaglobulinemia in chronic 
pancreatitis, celiac disease, and nephrotics as well 
as in two disorders previously described in idio- 
pathic hypercatabolic hypoproteinemia. A syn- 
drome including intestinal protein loss has 
been noted in six children with extreme eosino- 
philia and an allergic diathesis. Intestinal X-ray 
abnormalities were not significant. Intestinal 
biopsy examinations showed only eosinophilia of 
the mucosa. Two of these patients were given 
steroids and had a complete remission. Two addi- 
tional patients not treated with steroids responded 
completely, or in part, to elimination of dietary 
allergens, presumed to be milk proteins. In a 



NATIONAL CANCER INSTITUTE 



majority of the patients that were previously de- 
scribed as idiopathic, hypercatabolic hypoprotein- 
emia abnormalities of the intestinal lymphatics 
have been shown to be the major associated factor. 
This disorder of the lymphatics appears to be gen- 
eralized rather than limited to the intestinal tract 
since some of the patients had associated chylous 
effusions and lymphedema, or abnormalities of the 
lymphatic channels as shown by lymphangiog- 
raphy. The abnormalities demonstrated by lym- 
phangiography included the apparent absence of 
lymph nodes in the abdomen of one patient with 
an 11-year history of chylous ascities beginning at 
birth. ' 

The differentiation of gastrointestinal protein 
loss from failure of protein synthesis appears to be 
exceedingly important since in 23 of the 50 cases 
studied, there has been a reversal of the intestinal 
protein loss on appropriate therapy. Specifically, 
surgical resection has been shown to be of value in 
pericarditis and by others in patients with gastric 
carcinoma, giant rugal hypertrophy, and one case 
of intestinal lymphangiectasis. Corticosteroids 
have been shown to be of value in the treatment of 
celiac disease, regional ileitis associated with 
agammaglobulinemia, and the allergic disorder 
associated with eosinophilia, while oral antibiotics 
were of value in patients with Whipple's disease 
and agammaglobulinemia and a patient with 
chronic salmonellosis. 

Immunological Studies 

Two lines of laboratory work have been devel- 
oped. The first was the preparation of purified 
normal gamma globulins of each class, i.e., 6.6S 
gamma globulins, /? 2A globulins, and y^macroglob- 
ulins from normal serum and gammamicroglobu- 
lins from normal urine, as well as the purification 
of Type I and II Bence-Jones proteins, gamma- 
myeloma proteins, /J 2A -myeloma proteins and gam- 
mai-macroglobulins. The purified proteins have 
been used for a variety of metabolic and structural 
studies on gamma globulins. The purified proteins 
made possible a second line of laboratory work, i.e., 
the preparation of polyvalent antisera reacting 
with all gamma globulin groups and of specific 
antisera reacting only with 6.6S gamma globu- 
lins, /3 2A -globulins, gamma-macroglobulins and 
Type I or Type II gamma globulins. The availa- 
bility of specific antisera has made possible : 



(1) Simplified and more rapid diagnostic 
tests for multiple myeloma and macro-globu- 
linemia. 

(2) Identification and morphological char- 
acterization of the cells forming 6.6S gamma 
globulins, /?2A-globulins or yi-macroglobulin 
levels. 

(3) Quantitative methods for the specific 
measurement of serum 6.6S gamma globulin, 
/^-globulin, and yi-macroglobulin levels. 

Clinical studies in patients with macroglobu- 
linemia and multiple myeloma revealed both 
groups to have an increased susceptibility to bac- 
terial infection. The multiple myeloma patients 
had a significantly higher rate of infection than 
was found in the macroglobulinemic patients but 
no differences were found among myeloma patients 
forming gamma-myeloma proteins, /? 2A -myeloma 
proteins or Bence-Jones proteins. Both myeloma 
and macroglobulinemia patient groups were 
equally poor in their capacity to produce anti- 
bodies in response to antigen administration, and 
all had reduced levels of normal gamma globulin 
components reflecting generalized impairment of 
normal immune mechanisms. 

Further study of the usefulness of plasma- 
pheresis in the therapy of hyperglobulinemic hy- 
perviscosity syndromes revealed that arithmetic 
increases of serum macroglobulin level were ac- 
companied by logarithmic increases in serum vis- 
cosity. This laboratory evidence provided a 
theoretical basis for the rationale of intensive 
plasmapheresis in patients with macroglobulin- 
emic (or other) hyperviscosity syndromes. 

Nucleic Acid and Pyrimidine Metabolism 

Little knowledge was available until recently 
concerning quantitative aspects of human pyrimi- 
dine metabolism. This laboratory has been en- 
gaged in efforts to utilize urinary pseudouridine 
as a tool for investigation of pyrimidine metabo- 
lism, in a fashion somewhat analogous to uric acid 
in purine metabolism. 

The rate of pyrimidine production was esti- 
mated to be increased 2-3 fold in patients with 
chronic myelogenous leukemia. These patients 
were then given 6-azauridine and C 14 carboxyl- 
labelled orotic acid at various times after initia- 
tion of the therapy. By determining radioactiv- 
ity in respiratory C0 2 and urinary orotic acid it 



8 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



was shown that the rate of pyrimidine production 
was further increased several fold. The data sug- 
gest there may actually be an increase in the pro- 
duction of uridine nucleotides and that a block in 
conversion from orotic acid to ui'idine nucleotides 
might not be the only site of action of 6-azauri- 
dine. Gouty patients who were given labelled 
orotic acid showed no abnormality corresponding 
to the early purine shunt seen in this disease. 
C 14 -labelled orotidine was prepared and evidence 
obtained that it is metabolically inert in man. 

Urinary pseudouridine excretion was measured 
in patients under dietary control and compared 
with urinary uric acid and creatine. There was 
inconsistent creatinuria in both chronic mye- 
logenous leukemia and chronic lymphocytic leu- 
kemia patients. In agreement with reported data, 
the urinary uric acid levels were normal in patients 
with chronic lymphocytic leukemia and increased 
in chronic myelocytic leukemia. However, the 
chronic lymphocytic leukemia patients showed a 
very substantial elevation of urinary pseudouri- 
dine. Isotopic studies with orotic acid indicate 
this excessive pseudouridine excretion is a reflec- 
tion of generalized pyrimidine overproduction by 
these patients. Isotopic studies with uric acid 
further indicate that the low urinary uric acid is 
not caused by an increased degradation of uric 
acid. Measurements of acid soluble and insoluble 
nucleotide containing compounds of lymphocytic 
leukemia lymphocytes failed to indicate a chemi- 
cal preponderance of pyrimidines to account for 
the urinary findings. 

The enzyme systems converting thymidine mon- 
ophosphate to triphosphate have been investigated 
further. Although unusual mechanisms for this 
conversion have been suggested, our results tend 
to indicate that the conversion proceeds through 
the thymidine diphosphate and a separate enzy- 
matic activity is responsible for conversion to the 
triphosphate. 

An enzyme system incorporating uridine tri- 
phosphate into acid insoluble polynucleotide ma- 
terial from ribosomes has been identified. This 
material has been solubilized, partially freed of 
nucleic acid, and purified about 20-fold. Two 
additional enzymes from the cytoplasm of E. coli 
have been identified and separated. Each has been 
freed of the bulk of the contaminating nucleic 
acid and purified approximately 20-fold. One in- 
corporates guanosine triphosphate into polynucle- 



otide material, the other will incorporate uridine 
triphosphate into polynucleotide material in the 
presence of either DNA or UNA. 

In relation to studies on protein synthesis some 
purification has been achieved of the rather un- 
stable enzyme from mammalian cytoplasm of rat 
and rabbit (liver and thymus) converting guano- 
sine triphosphate through the diphosphate to the 
monophosphate. There is a possibility that this 
enzyme is related to the early stages of protein bio- 
synthesis but this situation remains to be clarified. 

Studies performed with HeLa cells indicate that 
under very precise control conditions, it is pos- 
sible to obtain a stimulation of uptake of radio- 
active phenylalanine into protein by exposing the 
cells to polyuridylic acid. 

Studies of Erythropoietin 

An improved and more sensitive assay system 
for erythropoietin utilizing radioiron incorpora- 
tion into red cells of the polycythemic mouse has 
been developed. Studies of erythropoietin have 
been directed toward the following : factors stimu- 
lating production of erythropoietin, its chemical 
nature, site of production, and metabolic rate. In 
mammals, bleeding, anoxia, and cobalt administra- 
tion are potent stimuli for erythropoietin produc- 
tion. The mechanisms of action of the fundamen- 
tal stimulus to erythropoietin production remains 
unknoAvn. Mice given intraperitoneal injections 
of homologous red cells and large quantities of 
dextran showed a simultaneous increase in the 
total red cell volume and reduction in the peri- 
pheral hematocrit and a marked depression of 
erythropoiesis. This suggests that the total body 
content of red blood cells rather than the concen- 
tration in the peripheral blood is a controlling fac- 
tor in erythropoietin production. In the frog 
bleeding of one-third the blood volume resulted 
in a marked stimulation of erythropoiesis. When 
frogs were maintained in a hypoxic atmosphere, 
there was no stimulus to erythropoiesis, indicating 
that in this species hypoxia is not a potent stimu- 
lus of erythropoiesis. The frog did not respond 
to cobalt or to human erythropoietin. However, 
the frog does produce an erythropoietinlike ma- 
terial since serum from anemic frogs contains a 
factor stimulating erythropoiesis in recipient 
frogs. 

The erythropoietin activity in the serum of 
anemic subjects from a cerebellar tumor and renal 



NATIONAL CANCER INSTITUTE 



cysts was nondialyzable, was not soluble in lipid 
solvents and was inactivated by trypsin and siali- 
dase. On electrophoresis the activity migrated 
with the alpha 2 globulins. The molecular weight 
was estimated at 25-30,000. The erythropoietic 
activity was neutralized by antibodies produced in 
the rabbit to a human erythropoietin extract. 
These studies indicate that these erythropoietins 
derived from different sources were similar. 

Measurable levels of erythropoietin were gen- 
erally found in patients with a hemoglobin con- 
tent less than 8 grams percent except for those 
with extreme renal damage. Erythropoietin was 
not found in patients with polycythemia vera or 
relative polycythemia. It was found in 50 per- 
cent of patients with polycythemia secondary to 
anoxia and in 30 percent of patients with poly- 
cythemia secondary to tumors. The rate of catab- 
olism of erythropietin in man was measured by 
determining the rate of decrease of erythropoietin 
activity following transfusions of aplastic anemic 
patients to hemoglobin levels sufficient to decrease 
erythropoietin production. In general, it has 
been found that at hemoglobin levels above 10 
grams percent that there is no detectable serum 
or urinary erythropoietin. When patients were 
transfused to this level, the half time of the rate 
of decrease of erythropoietin activity varied from 
11 to 35 hours in five patients, and in one patient 
was approximately 100 hours. Loss of erythro- 
poietin activity in the urine did not contribute 
to the decline in erythropoietin serum titer. 

The adrenal cortical tumor 494 carried in 
Osborne Mendel rat is associated with an increase 
in hematocrit in 60-70% and 100-200% increase in 
total circulating red cell volume. A saline extract 
of the tumor produced a small but statistically sig- 
nificant erythropoietic stimulation in the mouse. 
Administration of androgenic cortical steroids 
and an extract of normal adrenal gland did not 
produce such stimulation. 

Effects of Metabolic Rate on Erythropoiesis 

The effect on erythropoiesis of changes in the 
metabolic rate was studied in the dog, frog, and 
alligator. In the hypermetabolic dog, there was a 
25% increase in the total red cell volume and rate 
of red cell synthesis, but no change in the red cell 
life span. In the hypothyroid dog there was a 
40% decrease in the total red cell volume with a 
concomitant decrease in the rate of synthesis of 



the red cell, but no change in red cell life span. 
Thus in the dog the effects of changes in metabolic 
rate are in the rate of erythropoiesis and total red 
cell volume and not on the red cell life span. In 
poikilothermic animals, there was a significant al- 
teration in the red cell life span. In the frog 
taken from 20° to 4° C, there was an increase in 
the red cell life span from 125 to 250 days accom- 
panied by a considerable decrease in the rate of 
erythropoiesis. A similar change was seen in the 
alligator again Avith a marked decrease in the rate 
of erythropoiesis. 

Means of Measuring Red Cell Life Span 

Chromium 61 is widely used to measure red cell 
survival. The chromium content of the blood de- 
creases for two reasons: (1) elution of isotope 
from intact surviving cells; and (2) removal of 
senescent cells. Measurement of the rate of elu- 
tion of chromium in patients with hematologic dis- 
ease showed that this varied from 0.57 to 2.27% 
per day. This wide variation in the elution rate 
makes the use of Chromium 51 as a means of meas- 
uring red cell life span less satisfactory than 
isotopically labelled diisopropylfluorophosphate. 
The reticulocyte count was compared to the rate 
of production of red cells as measured both with 
radioactive iron and by measurement of the life 
span of the red cell. A coefficient of variation of 
0.70 was observed between reticulocyte count and 
rate of formation of red cells. However, in 
various specific instances, the deviation of the re- 
ticulocyte count from that predicted was large. 
It was concluded that the reticulocyte count re- 
mains a clinically useful tool but cannot be used 
to predict the rate of formation of red cells. 

Diisopropylfluorophosphate labelled with both 
P 32 and tritium can be used to measure the red 
cell lifespan. The use of the same chemical but 
with different isotopic markers permits the meas- 
urement of the survival of two populations of 
red cells and thus the differentiation of intra- from 
extra corpuscular factors leading to premature 
cell death. In the normal individual, the red cell 
life span of in vivo labelled and in vitro labelled 
donor cells was normal although the survival of 
the autochthonous cells was slightly longer than 
the homologous cells. Two types of variations 
were observed in disease. In some patients, the 
donor and recipient cells were removed at approxi- 
mately equivalent rates. In other patients, the 



10 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



recipient cells were removed at an accelerated rate 
while donor cells had a normal lifespan. In the 
former group there was some extracorpnscular 
factor leading to red cell shortening, and in the 
latter group, some intracorpuscular factor. 

Ineffective erythropoiesis can be defined as the 
production of red cells which are either destroyed 
in the marrow or very shortly after arrival in the 
blood. For some time it has been known that 
following the administration of isotopically la- 
belled glycine there was a peak of incorporation 
of isotope into bilirubin at 3 to 5 days, and again 
a second peak at 120 days. In the normal, the 
second peak represents the bilirubin derived from 
the catabolism of the hemoglobin in senescent cells. 
The source of the early peak was unknown, but 
thought to be associated with erythropoiesis. In 
two patients with failure to produce red cells, this 
early peak was almost entirely absent indicating 
that in large measure if not entirely, this early 
peak is associated with erythropoiesis. 

Porphyrins 

Studies of the biochemical lesions of porphyria 
in patients and in experimental animals have been 
continuing. The major goal of this project is 
to find the biochemical defect causing porphobili- 
nogenuria in both experimental animals and in 
human acute intermittent porphyria. Four major 
metabolic pathways have been studied which 
could, in theory, cause an increased excretion of 
porphyrin precursors. The only one of these 
which appeared to be involved in experimental 
porphyria was the oxidation of glycine to carbon 
dioxide. Glycine oxidation can occur by at least 
four well defined pathways: (1) conversion to 
delta aminolevulenic acid; (2) conversion to ami- 
noacetone; (3) conversion to serine, and (4) con- 
version to glyoxalate. Certain aspects of the first 
three pathways have been studied. 

The synthesis of ALA has been shown to occur 
in very small quantities in isolated mitochondria 
from normal liver. This activity is markedly in- 
creased in experimental porphyria. This path- 
way, therefore, cannot explain the decreased oxi- 
dation of glycine. Aminoacetone is formed from 
the condensation of glycine and acetyl CoA. The 
enzyme catalyzing this reaction is normally 
present in such quantity that the capability for 
formation of aminoacetone from glycine greatly 
exceeds the capability for formation of ALA. A 



deficiency of this enzyme in porphyria might ex- 
plain many of the chemical phenomena of porphy- 
ria. The amount of this enzyme present in the 
livers of porphyric rats is normal. Another source 
of aminoacetone which is probably a more impor- 
tant source than glycine is thereonine. This en- 
zyme pathway is not altered in experimental 
porphyria but is markedly decreased in the liver 
of tumor-bearing rats. 

In experimental porphyria, diet has a profound 
effect. This has been demonstrated in man by 
alteration in the excretion of amino-levulinic acid 
and porphobilinogen. The mechanisms involved 
are not known. Administration of estrogens in- 
creases porphobilinogen excretion in patients. In 
several female patients porphyria attacks could be 
related to the menstrual cycle. These patients 
have been treated successfully with estrogenic and 
androgenic materials. 

A continuing search for the mechanism of de- 
ceased amino-levulinic acid dehydrase in livers of 
tumor-bearing animals fails to reveal conclusive 
evidence of a material in tumors which can be ex- 
tracted and produce this change in recipient nor- 
mal animals. 

The mechanism of the lowering of liver catalase 
is different from that of lowering ALA dehydrase 
since "toxohormone" does not cause a decrease of 
ALA dehydrase and a decrease in catalase may 
precede that of ALA dehydrase. Injection of cor- 
tisone causes a decrease in hepatic ALA dehydrase 
activity and adrenalectomy causes an increase. 
The possibility that the decreased ALA dehyrase 
in the livers of tumor-bearing animals might re- 
sult from increased adrenal activity was investi- 
gated by growing tumors in adrenalectomized 
animals. Adrenalectomy did not prevent the de- 
crease of hepatic ALA dehydrase produced by 
tumors. The oxidation of thereonine to amino- 
acetone is profoundly decreased in the livers of 
animals bearing large tumors. 

DERMATOLOGY BRANCH 

The clinical and laboratory research activities 
of the Dermatology Branch continue to be con- 
cerned with two major areas; namely, (1) study 
of normal and abnormal growth and differentia- 
tion of the epidermis and related epithelium and 
(2) study of the lymphomatous disease mycosis 
fungoides. 



NATIONAL CANCER INSTITUTE 



11 



1. Epidermal Growth and Differentiation 

Recent work in this Branch has established that 
mitotic division of epidermal cells is normally 
confined to the single layer of cells in direct con- 
tact with the underlying connective-tissue corium. 
In vivo studies measuring the time of appearance 
of C 14 -glycine in proteins shed at the skin surface 
have indicated that the epidermal turnover time is 
28 days. Since the epidermis is approximately 28 
cell layers hi thickness, the inference is made that 
the basal cell layer reduplicates itself one- fold each 
day, and that the transit time of a newly-formed 
cell, from its site of production, to the epidermal 
surface is also approximately 28 days. During its 
transit at least three distinct fibrous (keratinous) 
proteins are produced, each at a different level 
within the epidermis since these proteins, labelled 
with C 14 -glycine reach the surface at different 
times. (See Project #3602, 1962.) 

In psoriasis, the germinative basal-cell popula- 
tion has been found to be increased to three cell 
layers, and is further increased by another factor 
of 3 by folding of the epidermal-dermal line. Thus 
the theoretical transit time of newly formed cells, 
from the basal zone to the epidermal surface in 
psoriasis may be mathematically calculated to be 
3-4 days. In vivo studies, measuring the time of 
appearance of C 14 -glycine-labelled proteins at the 
epidermal surface confirms this. The epidermal 
hyperplasia in psoriasis therefore seems mainly 
due to a ninefold expansion of germinative basal 
cells, rather than due to increased mitotic rate of 
a fixed number of basal cells. Faulty maturation 
of epidermis in psoriasis seems secondary to 
marked decrease in time allowed for maturation of 
cells before they are shed. 

In the normal hair root, the entire root matrix is 
comprised of germinative cells, which in large scalp 
hairs may constitute as many as 30 cell layers. 
Daily reduplication of this large cell population 
accounts for the rapid growth "rate" of hair. 

Further studies in this Branch indicate that a 
basal cell tumor arises because of inability of its 
constituent cells to produce normal fibrous keratin 
proteins. A major protein, found in normal 
epidermis, is not found in this tumor, a finding 
corroborated by our earlier studies and recently by 
an electron microscopic study from another institu- 
tion which has shown the absence of structural 
fibrous proteins in basal cell tumor cells. A basal 



cell tumor hence forms from basal cells which are 
unable to pass through the normal epidermis via 
the process of keratinization, and which, by retain- 
ing their normal capacity for mitotic division, en- 
large into ever increasing masses below the 
epidermis. 

The role of normal connective tissue environ- 
ment in maintaining normal biologic behavior of 
epithelial cells, and the influence of altered en- 
vironments, have been further analyzed. Human 
epidermal cells, removed from their normal en- 
vironment and cultured in vitro (Laboratory of 
Biology, NCI) appear to have lost normal behav- 
ior capabilities (or gained abnormal capabilities). 
Freshly removed epidermis or epithelium, auto- 
implanted to new sites in the integument, responds 
in predictable normal patterns. Implanted cul- 
ture cells, on the other hand, reveal no evidence 
of differentiation into any of the normally ex- 
pected patterns and have histologic appearances 
of undifferentiated cancer cells. 

2. Mycosis Fungoides 

Evaluation of the status of immunologic reac- 
tivity of patients with mycosis fungoides has 
continued. 

Skin tests with a series of antigens have demon- 
strated that a significant depression of delayed 
sensitivity is characteristic of mycosis fungoides 
only late in the course of the disease. The homo- 
graft rejection mechanism is intact. In patients 
who have well-defined areas of cutaneous involve- 
ment a provocative pattern of acceptance and 
rejection of autografts has been noted. Normal- 
to-normal, involved-to-involved, and normal-to- 
involved transplants take in the usual fashion. 
Approximately 75% of involved-to-normal trans- 
plants are rejected in what appears to be a typical 
delayed reaction characteristic of the response seen 
to homografts. 

A horse has now been injected 15 times with 
homogenized tumor from 10 patients with mycosis 
fungoides. Serum from the horse has a band on 
Ouchterlony plate against mycosis fungoides 
tumor tissue. There is also a band against serum 
from normal patients and to serum from patients 
with mycosis fungoides. Complement fixation 
tests against mycosis fungoides tissue from three 
different patients are posistive; normal skin does 
not absorb out this antibody with techniques em- 
ployed thus far. 



12 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



When the horse serum is run against normal 
human serum in immunoelectrophoresis there is a 
good band to albumin, alpha 2 and beta globulins 
and a poor band to gamma globulins. 

Three patients with mycosis fungoides have re- 
ceived intratumoral injections with this serum. In 
each there has resulted necrosis of tumor at the 
site of injection. No such reaction occurs in un- 
involved skin of these patients when injected with 
the serum, nor in normal skin of patients without 
mycosis fungoides. 

ENDOCRINOLOGY BRANCH 

This year has permitted the Endocrinology 
Branch to fully set up an independent research 
program for two recently acquired senior investi- 
gators. Moreover, these individuals have been 
effectively integrated into our patient-care pro- 
gram in such a way that each of our senior investi- 
gators now accept major clinical responsibilities 
for only 4 months at a time and then has 8 consecu- 
tive months for continuous research effort. Mean- 
while, the overall coordination of clinical and re- 
search functions continues to be worked out 
through daily informal discussions and through 
our weekly grand rounds. 

It is gratifying to note that whereas only one of 
last year's clinical associates elected to remain for 
a third year, two of this year's group have already 
requested a third year in research. We anticipate 
that these third year men will greatly enhance 
our research effort in coming years and will pro- 
vide potential recruits for future staff positions. 

Two highly practical accomplishments in the 
field of the chemotherapy of choriocarcinoma and 
related trophoblastic tumors in women seem to 
stand out for this year. The first is the successful 
application of chemotherapy as a primary form 
of treatment in nonmetastatic trophoblastic dis- 
ease. It may be anticipated that thousands of 
young women in coming years may be spared the 
loss of their reproductive capacity by this form of 
nonsurgical treatment. Moreover, in principle, 
this represents the first instance of the substitu- 
tion of chemotherapy for surgery in the effective 
and definitive management of malignant disease. 

The second noteworthy development is the sta- 
tistically valid establishment of the effectiveness 
of Actinomycin D in the methotrexate-resistant 
patients, thus increasing the expected complete re- 
mission rate from 47% to 76%, as manifested by 



the last 38 patients with metastatic trophoblastic 
disease admitted for study. 

At a more basic level, the studies of our steroid 
group have provided exquisitely sensitive tools for 
the isotopic analysis of the metabolic behavior of 
numerous vital steroid substances whose role in 
malignancy and in related disease processes has 
hitherto remained obscure. This newer meth- 
odology may be expected to elucidate many of the 
problems in this area. 

The development of isotopic methods for trac- 
ing one of the most potent of biologically active 
trace elements, namely biotin, may also be re- 
garded as a significant methodological accomplish- 
ment in a most difficult area of immediate perti- 
nence to the cancer problem. 

Basic studies on the endogenous and exogenous 
factors involved in hormone-induced tissue growth 
provide essential background data for new ap- 
proaches to the control of normal as well as neo- 
plastic growth in hormone-sensitive tissues. The 
development of newer pharmacologically active 
agents which can alter such growth processes con- 
tinues to demand our best efforts. 

On the whole, it may be concluded that the free 
pursuit of basic information combined with a 
readiness to exploit such data in a practical man- 
ner may be expected to provide research develop- 
ments of value and interest. 

MEDICINE BRANCH 

A comprehensive nationwide approach to the 
study of the treatment of acute leukemia has been 
undertaken by the Leukemia Task Force. The em- 
phasis as regards chemotherapy is in the follow- 
ing areas : the rapid introduction into the clinic 
of agents which show significant activity in trans- 
planted rodent tumor systems; the effective per- 
formance of Phase I therapeutic trials in acute 
leukemia ; quantitative clinical trials to determine 
the relative efficacy of agents as regards their abil- 
ity to induce and maintain remissions ; and a com- 
prehensive analysis of animal screens to determine 
which correlate most closely with the clinical data. 
The Medicine Branch has participated in a major 
way in the planning, execution, and evaluation of 
a number of these studies. 

New agents which have proven effective in the 
treatment of acute leukemia during the past two 
years include vincristine, Cytoxan, and guanyl- 
hydrazone. Vincristine produces remissions 



NATIONAL CANCER INSTITUTE 



13 



rapidly in 50-60% of children with acute lympho- 
cytic leukemia. A coded study wherein vincris- 
tine is compared to a placebo in the maintenance 
of vincristine induced remissions is nearing com- 
pletion. Preliminary analysis would suggest that 
vincristine is not effective in maintaining remis- 
sion (Karon). Guanylhydrazone produces com- 
plete remissions in 30-50% of patients with acute 
myelogenous leukemia (Freireich). The toxicity 
of this agent limits its usefulness. Attempts to 
improve the therapeutic index are underway but 
have, to date, not proven successful. These in- 
clude (1) combinations with other agents, 

(2) modification of route and dose schedule, and 

(3) study of congeners of guanylhydrazone. (See 
below.) 

Pharmacologic and animal tumor studies indi- 
cate that the schedule of Methotrexate (MTX) 
administration to man should be important in 
achieving maximum therapeutic effect. In 21 pa- 
tients with acute leukemia, 18 (86%) achieved re- 
mission with MTX administered every 4 days as 
compared to 30% for conventional daily adminis- 
tration (Freireich). 

New agents currently under study in acute leu- 
kemia in the Medicine Branch include hydroxy- 
urea, the terephthalanalides, CB-1506, nitroso- 
urea, azauridine, and etiocholanalone. 

During the past several years the Medicine 
Branch has increased its program in the study of 
patients with chronic leukemia, lymphoma, and 
myeloma and has decreased the number of beds 
allotted to patients with "solid" tumors. In order 
to continue our program in the solid tumor area, 
a 15-bed chemotherapy unit has been established at 
the USPHS Hospital in Baltimore under Dr. N. 
Tarr, their Chief of Surgery, and the Medicine 
Branch, NCI. 

There are a number of agents of established 
efficacy in chronic myelogenous leukemia (CML). 
New agents which we have found to be effective in 
these patients include azauridine, hydroxyurea, 
and vinblastine (Carbone). One of the most im- 
portant problems in the treatment of CML is that, 
though a number of agents are capable of induc- 
ing and maintaining remissions, survival has not 
been significantly prolonged. In an effort to de- 
fine further this problem, an analysis of the effects 
of treatment with conventional agents (6-mercap- 
topurine, Myleran, colcemid) on the hematologi- 
cal, biochemical and cytogenetic abnormalities in 



these patients has been undertaken. The cyto- 
genetic aspects of this problem were particularly 
important because a chromosomal marker, the 
Philadelphia chromosome, has been observed in 
the majority of patients with CML and does not 
occur in other neoplastic diseases. It has been 
found that 90% of patients will have effective re- 
duction of the white blood cells and organo- 
megaly; 40-50% of patients will in addition have 
return of their bone marrow to "normal"; 40% of 
patients will have return of the white cell alkaline 
phosphatase to normal ; but in no patient, regard- 
less of the extent or duration of remission, does the 
Ph' chromosome disappear from the marrow. Al- 
most invariably 100% of marrow metaphases have 
the chromosome prior to treatment and no reduc- 
tion occurs in spite of otherwise effective treat- 
ment. If the Ph' chromosome marks the malig- 
nant cell it is clear that the above treatment is only 
palliative, i.e., the majority of the leukemic cells 
are destroyed but some always remain. The im- 
plication of this as regards experimental therapy 
of this disease will be discussed (see below) 
(Block, Carbone). 

New agents recently found to be active in 
Hodgkin's disease and lymphosarcoma include 
vincristine, vinblastine, and guanylhydrazone. 
The first two produce regressions in 50-80% of 
these patients and thus compare with the alkylat- 
ing agents. Preliminary studies indicate that they 
are not cross-resistant clinically with each other 
or with the alkylating agents (Carbone). Remis- 
sions produced with guanylhydrazone are often 
complete but transient and occur at considerable 
cost in toxicity. The fact that there are now a 
number of effective therapeutic modalities for the 
lymphomas (X-ray, alkylating agents, periwinkle 
alkaloids, corticosteroids, guanylhydrazone, and 
methotrexate) indicate that a more comprehensive 
and disease oriented approach to therapeutics is 
desirable. 

Most of the Medicine Branch studies in patients 
with "solid" tumors hahve been performed in col- 
laboration with the Eastern Solid Tumor Group. 
Comparative studies of Cytoxan, uracil mustard, 
and nitrogen mustard have failed to show signifi- 
cant differences for these agents in patients with 
solid tumors or lymphomas. A coded study of 
fluorouracil, fluorodeoxyuridine, and methotrex- 
ate in patients with carcinoma of the breast and 
colon indicate some differences between these 



14 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



agents in terms of therapeutic index. Prelimi- 
nary studies of vincristine, vinblastine, guanyl- 
hydrazone, hydroxyurea, etiocholanolone, nitro- 
surea, and tryptophan mustard have shown 
limited activity for vincristine and vinblastine in 
cancer of the breast and suggestive activity in cer- 
tain other solid tumors (Perry, Carbone*. 

Phenylalanine mustard affords objective benefit 
to 30% of patients with multiple myeloma. In a 
coded study performed by the Eastern Solid 
Tumor Group, there was no difference between a 
placebo and urethane in 100 patients with multiple 
myeloma. In this study objective improvement of 
any sort occurred in less than 5% of the patients. 
This study affords considerable information con- 
cerning the nautral history of myeloma and thus 
serves as a frame of reference for future studies. 

In addition to the above, studies involving the 
following therapeutic approaches are underway : 

1. Remission Induction 

Since complete remissions can now be induced 
with a number of agents in the acute leukemias, 
lymphomas, and chronic leukemias, combinations 
of these effective agents used intensively during 
remission, induction and early maintenance might 
be curative. Preliminary studies of vincristine, 
prednisone, methotrexate, and 6-mercaptopurine 
used in combination in patients with acute leuke- 
mia, and combinations of nitrogen mustard and 
vincristine in patients with lymphoma have 
yielded promising results (Frei, Freireich, Rail, 
Carbone) . 

2. Remission Maintenance 

The development of resistance of malignant 
cells to chemotherapeutic agents is a major prob- 
lem and limits current clinical cancer chemother- 
apy. Approaches to control of this problem in- 
clude combination chemotherapy studies involving 
the use of MTX and 6-mercaptopurine both con- 
currently and alternating at monthly intervals in 
maintaining acute leukemia remissions. Prelimi- 
nary analysis suggests that the alternating main- 
tenance therapy does not prolong remission. The 
sequestration of leukemic cells in areas not accessi- 
ble to antileukemic agents (brain and meninges, 
and questionably, thymus) may serve as a focus 
for the development of the resistant cell. In a 
controlled study of remission maintenance the 
use of intrathecal aminopterin prophylactically 



throughout the remission has failed to prolong re- 
missions (delay the development of resistance) 
(Frei). Autopsy studies of patients with menin- 
geal leukemia indicate that, though intrathecal 
aminopterin markedly reduces the number of 
leukemic cells on the meninges, it does not eliminate 
them (Thomas). The nitrosourea derivatives are 
active against intracranial as well as subcutaneous 
L-1210 leukemia in mice. This drug has physioco- 
chemical properties consistent with distribution 
into the central nervous system. Finally, pre- 
liminary studies in man indicate that orally ad- 
ministered nitrosourea is effective in treating 
meningeal leukemia. 

Cytogenetics 

The specificity of the Ph' chromosome marker 
for the chronic myelogenous leukemia (CML) cell 
is established and 90+% of patients with CML 
have this change (Carbone, Whang) . It is present 
not only in myeloid tissue but also in nucleated 
red blood cells and probably megakaryocytes 
(Whang). If the Ph' chromosome marks the 
neoplastic cell then all three cellular elements of 
the marrow are malignant and presumably the 
neoplastic change occurs in a stem cell. Attempts 
to identify this cell are underway. Cytogenetic 
studies of acute leukemia have yielded varying but 
inconsistent patterns from patient to patient. 
Within the same patient, however, cytogenetic ab- 
normalities, if present, persist and tend not to 
change (Whang). 

Cytogenetic techniques have proven most useful 
in following tissue homografts. Homologous 
CML cells transfused into patients with acute 
leukemia (see below) have persisted and .replicated 
in the recipients for as long as 60 days as evi- 
denced by the presence of the Ph' marker. Most 
important is the fact that in some of the recipients 
the grafts functioned in terms of maturation and 
delivery into the peripheral blood of granulocytes 
and probably platelets and red cells. Skin graft 
studies have not shown tolerance to CML donor 
skin and overt evidence of homologous disease has 
not occurred (Levin, Whang, Freireich, Frei). 

Marked improvement has been achieved in the 
prevention of thrombopenic hemorrhage by ho- 
mologous platelet transfusions. Complement 
fixing antibodies develop and limit donor platelet 
effectiveness in less than 15% of patients and iso- 
antibodies do not decrease platelet survival. Ho- 



NATIONAL CANCER INSTITUTE) 



15 



moloo-ous CML white cells at doses in excess of 10 11 
control major infections, including pseudomonas 
septicemia in the majority of instances. Antibody 
formation limiting white blood cell survival in 
patients with acute leukemia has not been a major 
problem (Freireich) . In order to expand this re- 
search and extend its clinical usefulness methods 
for the more effective acquisition and storage of 
white cells and platelets are being developed. A 
centrifuge is under development, which should 
allow for immediate return of red blood cells and 
plasma to the donor and continuous separation of 
the white cells and platelets (Judson, Freireich). 
This would be far more effective than the currently 
used batch plasmapheresis and would allow for 
the use of normal white cells rather than the white 
cells from patients with chronic leukemia. 

In view of the above studies we have intensified 
our efforts to define more precisely the effect of 
cancer chemotherapeutic agents on host defense. 
In a controlled study of 6-mercaptopurine in man 
it has been found that this agent depresses circu- 
lating antibody response to primary but not to 
booster antigenic stimulation, that it partially de- 
presses skin homograft rejection, that it does not 
decrease established delayed skin sensitivity but 
does suppress the induction of delayed skin sensi- 
tivity (Levin, Frei). Similar studies are being 
performed with relation to other cancer chemo- 
therapeutic agents. 

PHARMACOLOGY AND RELATED STUDIES 

The blood-brain barrier is of major importance 
to pharmacology generally and to cancer chemo- 
therapy particularly. Tumors, both primary and 
metastatic frequently develop within the central 
nervous system. Most cancer chemotherapeutic 
agents do not appear to pass the blood-brain bar- 
rier. Increasing knowledge of the physiocochemi- 
cal properties which influence passage across the 
blood-brain barrier should markedly improve 
treatment. Finally, the problems of cell entry and 
passage across the blood-brain barrier have many 
things in common and it may well be that the cen- 
tral nervous system acts as a typical cell and that 
penetration there and penetration into other cells 
are fundamentally similar. 

Using perfusion techniques the rate of produc- 
tion of spinal fluid in the dog has been determined 
as has the rate of removal of bulk flow. The 



amount of extracellular space in the brain has 
been the subject of considerable controversy, the 
major evidence being based on electron micro- 
scopy which indicated very little extracellular 
space. Using the above techniques and C 14 -inulin 
it has been determined that the extracellular space 
in the brain is of the order of 10 to 15% of the 
brain by weight. When similar infusions are 
done in animals after death, the extracellular space 
is 2 to 6% similar to that estimated from electron 
microscopy studies (Rail). Many substances ap- 
peared to be removed from the cerebral spinal 
fluid passively. Recent evidence indicates that an 
active mechanism for removal of magnesium ex- 
ists (Rail, Oppelt). 

Studies of the pharmacology, enzymatic and 
biological effects of various analogs of folic acid 
antagonists continues. Using chlorine 30 labelled 
dichloromethotrexate, it has been found in ani- 
mals that absorption from the gastrointestinal 
tract is incomplete, that a major portion is ex- 
creted via the liver, that the compound does not 
enter the central nervous system, and that de- 
pending upon the species, a considerable portion 
of the dichloromethotrexate is hydroxylated to a 
much less active metabolite. The relative ability 
of various species to hydroxylate dichlorometho- 
trexate explains in part the difference in toxicity 
between dichloromethotrexate and methotrexate 
which is not inactivated. The markedly superior 
antileukemic activity for dichloromethotrexate as 
opposed to methotrexate does not occur in man, 
and has not been explained (Davidson, Oliverio, 
Adamson). Tritiated methotrexate has been pre- 
pared and pharmacologic studies of this agent are 
under way (Oliverio). The hydroxylating en- 
zyme for DCM has been studied in liver homog- 
enates and has been found to be present also in 
the histologically mature Morris hepatoma 
(Adamson). The oxidative produce is 7-hy- 
droxy-dichloromethotrexate which inhibits folic 
acid reductase some 100-fold less than does DCM 
itself (Oliverio, Misra). Seven-hydroxy amino- 
pterin, 7-hydroxy methotrexate, 7-isoaminopterin, 
7-iso folic acid, 7-methyl aminopterin and 7- 
methyl folic acid have been synthesized (Loo). 
These compounds show no inhibitory activity 
against L-1210 and only minimal inhibitory effect 
on frolic acid reductase in vitro (Loo, Adamson, 
Misra) . 



16 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Iii collaboration with the CCNSC a number of 
congeners of methylglyoxal-bis-guanylhydrazone 
have been synthesized and studied for activity 
against L-1210. Almost any deviation from the 
parent molecule results in considerable or com- 
plete loss of antileukemic activity (Davidson, 
Bond). C 14 methylglyoxal-bis-guanylhydrazone 
has been synthesized and its pharmacology in 
animals and to a limited extent in man has been 
studied. In rodents excretion is reasonably 
prompt, while in dogs, monkeys and particularly 
in man this compound, after a single dose, is ex- 
creted for many days (Oliverio). Methylglyoxal- 
bis-guanylhydrazone sensitive L-1210 leukemia 
concentrates C 14 methylglyoxal-bis-guanylhydra- 
zone 5-fold greater than does methylglyoxal-bis- 
guanylhydrazone-resistant L-1210 (Adamson). A 
chemical method for the analysis of hydroxyurea 
has been developed and pharmacologic studies 
using this method have been performed in both 
animals and man (Davidson). Toxicologic 
studies with a number of the terephthalanilides 
have been performed. Of interest is the fact that 
in monkeys most of these agents produce degen- 
erative changes in the extraocular muscles of the 
eye. This has served as a useful predicting sys- 
tem for those which produce ophthalmoplegia in 
man (Rail). 

The serotonin-producing mast cell tumor has 
been studied extensively as regards the effect of 
various chemotherapeutic agents. The best of 
these which include Cytoxan and the glutamine 
analogs, were selected for trial in patients with 
serotonin producing carcinoid tumors. In both 
mice bearing the mast cell tumor and in humans 
with the carcinoid tumor a marked increase in 
serotonin or urinary indoles occurs following 
treatment with either glutamine analogs or Cy- 
toxan (Kelly). Whether this represents tumor 
destruction in man is unknown. The various 
plasma cell tumors in mice are being studied ex- 
tensively with several chemotherapeutic agents. 
The antitumor effect of chemotherapeutic agents 
does not vary with the different forms of plasma 
cell tumors, many of which have qualitatively dif- 
ferent protein abnormalities (Carbone). 

Monkeys are being used increasingly for phar- 
macologic as well as numerous biological studies. 
The care and breeding of these animals is receiving 
major attention and considerable progress has 
been made during the past 18 months. Chemical 



carcinogens have been studied largely in rodents. 
A number of studies are underway concerning the 
ability of these chemical carcinogens to produce 
tumors in primates, particularly newborn monkeys 
(Kelly, Rail). 

The use of tissue culture as a tool to support bio- 
logical studies in a number of areas is increasing. 
The relative effects of bromodeoxyuridine on dif- 
ferent cells in cell culture correlates well with the 
relative ability of nucleosides to enter these cells. 
Preliminary success has been achieved in the cul- 
ture of human chronic myelogenous leukemic cells 
bearing a chromosome marker which can be iden- 
tified in culture (Mohler). 

BIOCHEMICAL STUDIES 

A number of biochemical studies, particularly 
of the human leukemic cell, are underway. Sev- 
eral of these relate to changes induced by the ad- 
ministration of cancer chemotherapeutic agents 
and their relation to response and resistance to 
such agents. Hypoxanthine and guanine analogs 
must be converted to the nucleotide by the enzyme 
inosinic pyrophosphorylase before their antileu- 
kemic effect can be exerted. Deletion of this en- 
zyme has regularly occurred in animal tumor sys- 
tems with drug induced resistance. Studies in 30 
patients with acute leukemia have indicated that 
major changes in this enzyme level in the acute 
leukemic cell do not occur and that resistance in 
the majority of patients to 6-mercaptopurine 
probably has another explanation (Davidson). 
In tissue culture studies it was shown that the 
variation in resistance to azaguanine may be as 
much as 100-fold in the presence of essentially 
complete inosinic pyrophosphorylase deletion. 
Clearly factors other than deletion of this enzyme 
are operative in the development of 6-mercapto- 
purine resistance (Davidson, Law). The initial 
effects of azauridine in the treatment of acute and 
chronic leukemia are excellent but resistance 
rapidly occurs precluding the development of re- 
mission in acute leukemia. Preliminary evidence 
would indicate that this resistance relates to a 
marked inductive increase in orotodylic decar- 
boxylase or orotodine pyrophosphorylase (Bono). 
Studies of tritiated thymidine have indicated 
varying incorporation into DNA for different 
types of white cells which correlates poorly with 
biological observations concerning the prolifera- 
tive capacity of these cells. Since thymidine ki- 



NATIONAL CANCER INSTITUTE 



17 



nase rather than DNA synthesis may limit incor- 
poration, the assay of this enzyme in various types 
of leukemic and normal white cells in parallel with 
studies of tritiated thymidine uptake is underway 
(Perry). 

By studying the relative intra- and extra-cellu- 
lar distribution of a weak non-protein bound acid 
it has been possible to determine intra-cellular pH. 
This technique plus an assay of intra-cellular acid 
production has been applied to normal and leu- 
kemic cells. A consistent gradient between intra- 
cellular and extra-cellular pH has been found for 
normal white cells which rapidly disappear fol- 
lowing injury of these white cells. The intra- 
cellular pH is more acid than the extra-cellular 
pH at almost all levels of extra-cellular pH below 
7.4. This pH gradient is considerably decreased 
in human leukemia cells and in at least some 
patients with chronic myelogenous leukemia re- 
turns to normal following effective treatment 
(Block, Rail). 

Using techniques for the detection of DNA de- 
naturation it has been observed that nitrogen mus- 
tard in very low concentration prevents denatura- 
tion by any of a number of denaturing agents. 
This is presumably the result of cross-linking and 
even concentrations of nitrogen mustard which 
produce very few cross links are capable of this. 
It has been shown that such DNA may induce ge- 
netic transformation whereas comparable DNA 
similarly denatured but not exposed to HN 2 loses 
this biological property. These techniques allow 
for a number of fundamental observations con- 
cerning nucleic acids and the effects of various 
drugs thereon (Kohn). Chromatographic anal- 
ysis of normal and leukemic white blood cell RNA 
has revealed distinct differences in the patterns 
for the malignant cells which resemble those of 
other immature cells. There is some evidence 
that this difference may result at least in part from 
phenol-resistant ribonuclease activity rather than 
a difference in the original RNA (Karon). 

RADIATION BRANCH 

The objectives of the Radiation Branch are to 
introduce and to maintain high standards of 
clinical cancer radiotherapy and nonclinical ex- 
perimental and service irradiations, to engage in 
supportive physics dosimetry and development 
programs, to study radiation-induced aberrations 
of structure and function, to develop biological 

705-685—63 3 



bases for the testing of new theoretical concepts 
which may be introduced into clinical practice, and 
to introduce such new concepts into applied clinical 
cancer radiotherapy as have been demonstrated in 
biological or other models as having potential 
value. 

The study of radiation-induced aberrations of 
structure and function is a continuous operation 
which is carried on whenever a patient is submitted 
to cancer radiotherapy and then followed. Im- 
portance is attached to both the anticancer and the 
normal tissue and organ responses to irradiation. 
Such studies may be general and comprehensive in 
relation to the specific anticancer radiotherapeutic 
effort which is being undertaken or the irradiation 
procedure may be designed around the specific 
study which it is intended to perform. In the 
former category are the single dose irradiation, the 
metabolic balance, and the childhood cancer studies 
and in the latter the renal function and the blood 
cell kinetics studies. 

The laboratory of radiobiology of the Radiation 
Branch has completed a definitive study of the 
relationship of ionization density or linear energy 
transfer (LET) to the oxygen effect which pro- 
vides an experimental foundation for the intro- 
duction of advanced theoretical concepts of accel- 
erated particle irradiation into clinical radio- 
therapy practice. The laboratory is now adapting 
the DBA-P-388 model into a completely pharma- 
cological mammalian in vivo quantitative tumor 
cell system which will permit the simultaneous 
experimental determination of the supplemental 
relationships and their mechanisms between phar- 
macological agents and irradiation in respect to 
both their antitumor effects and drug toxicity. 
Other studies of the DBA-P-388 systems have 
demonstrated that under certain conditions of host- 
tumor relationships the oxygen diffusion gradi- 
ents and the membrane transport are physiological. 
Preliminary studies of what is tentatively identi- 
fied as a transplantable chondrosarcoma of the 
Street strain mouse suggest possible experimental 
approaches to making more effective use of S-35 
in the treatment of human chondrosarcoma. 

SURGERY BRANCH 

The Surgery Branch of the National Cancer In- 
stitute continues to act in a dual capacity, that of 
carrying out its own specific clinical and labora- 



18 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tory investigations, and that of providing consult- 
ative surgical service to the National Institutes of 
Health. There were 1,068 requests for surgical 
consultation and 439 operative procedures were 
performed on these patients. This consultation 
service, in many instances, provided investigators 
with human tissue, both benign and maligant, for 
their research activities. 

The major clinical investigative interests of the 
Branch are directed to the indications for and the 
effectiveness of an aggressive surgical approach 
to cancer. Patients so treated have afforded the 
investigator an opportunity for lifetime observa- 
tions on the behavior of cancer as it is modified by 
therapy. 

Local wound recurrence of tumor, often re- 
ferred to as "wound seeding", is being studied 
both clinically and in the laboratory. Although 
we have found no experimental agent to control 
wound seeding, unless it be proflavine hemisulf ate, 
it has been demonstrated that the mode of appli- 
cation of a potentially therapeutic agent is most 
important. The growth of tumor in an artificially 
"tumor seeded" animal wound is directly propor- 
tional to the pressure by which the tumoricidal 
agent is applied. 

There continues to be a 26-30% incidence of 
wound washings positive for cancer cells follow- 
ing definitive surgery and a much lower but cor- 
related incidence of cytologically recognizable can- 
cer cells in the wound drainage during the first 
few postoperative days. 

Patients with carcinoma of the cervix continue 
to show an overall 40% 5-year survival follow- 
ing extensive surgery for advanced, usually re- 
current, disease end an operative mortality of less 
than 10%. 

Plastic reconstruction of the vagina following 
its total extirpation for cancer has been carried 
out on 10 patients. Six of these have been fol- 
lowed for 6 months and have functionally satis- 
factory vaginas. 

Both a short- and long-term evaluation indi- 
cates that the use of the ileal conduit following 
total pelvic exenteration is the most satisfactory 
method of urinary diversion. Although many of 
these patients have an asymptomatic course in 
spite of persistently positive urine cultures, the 
incidence of pyelonephritis is significant. Anti- 
bacterial prophylaxis has been of definite help in 
minimizing this complication. 



An evaluation of abdominal wound closure using 
wire in a single layer peritoneal fascia approxi- 
mation has shown that wound dehiscence can be 
prevented and wound herniation minimized. The 
development of a hernia or delayed suture abscess 
is directly correlated with the incidence of postop- 
rative wound infection. 

Of six postoperative hernias, five were in pa- 
tients who had infection. Delayed abscesses (up 
to 1 year) were not seen in patients who had had 
wound infection primarily. 

Bacteriological examination of all patients has 
shown that there is an increased postoperative in- 
fection rate in hemolytic staphylococcus aureus co- 
agulase positive carriers. Ten days of postopera- 
tive antibiotic therapy lowered the infection rate 
from 54.3% in a nonantibiotic treated group to 
14% in a treated group. When antibiotics were 
administered for 3 days preoperatively and 7 days 
postoperatively, the infection rate was 8% as com- 
pared to 17.4% in the patients who received only 
7 days of postoperative therapy. A 2 year survey 
has shown that when extensive and prolonged 
surgical procedures are undertaken, antibiotics are 
of definite value in decreasing the number of in- 
fectious complications. 

Lymphangiography has been performed on over 
100 patients and its usefulness has been demon- 
strated. This technique has been of considerable 
value in demonstrating the variability of the tho- 
racic lymphatic duct. A characteristic picture of 
incomplete lymph node filling by the injected dye 
is seen in metastatic disease from solid tumors. 
Large nodes with a foamy reticular pattern usu- 
ally indicate lymphomatous disease. The addi- 
tion of chlorophyll to the injectable radiopaque 
material stains the lymph nodes green and has sig- 
nificantly improved the completeness of lymph- 
adenectomies. 

Elective esophagostomy has been extensively 
used for intubation for feeding and has proven 
to be far superior to gastrostomy or an indwelling 
nasal tube. 

The double blind preoperative radiation study 
of head and neck cancer patients has continued to 
be an interesting project. One thousand roentgens 
given to the tumor area 24 hours preoperatively 
probably has caused some increase in morbidity, 
but the followup time is too short to determine 
any effect the radiation may have on the end 
results. 



NATIONAL CANCER INSTITUTE 



19 



Cancer cells may be present in the circulating 
blood of patients with cancer. The presence of 
circulating tumor cells cannot be correlated with 
survival. We have shown that present methods 
of recovering cancer cells are much more inefficient 
than had been suspected. Present efforts are di- 
rected at developing methods of tumor cell re- 
covery which will prove to be more efficient. It 
is suspected that circulating megakaryocytes may, 
in the past, have been incorrectly designated as 
tumor cells. 

Several patients have now been intensively stud- 
ied with the cooperation of the Metabolism Service 
in relation to metabolic alterations incidental to 
preoperative bowel preparation. Initial studies 
indicate a rather marked loss of fluid and electro- 
lytes and negative nitrogen balance. It is hoped 
that as a result of this study we may be able to 
ameliorate, in part, the metabolic alterations 
brought about by bowel sterilization. 

Survival from paranasal sinus cancer, in spite 
of improved techniques of radiation and surgery, 
continues to be very poor, usually due to local re- 
current disease. The Surgery Branch continues 
its active interest in cancer of this anatomical area 
and have combined a neurosurgical approach to 
the usual surgical approach in 23 patients. With 
elevation of the frontal lobe, intracranial invasion 
can be ruled out and the paranasal sinus area freed 
from the intracranial approximation. This allows 
en bloc resection of the entire area from the usual 
facial approach. 

In wound-washing studies whereby an open 
wound is seeded with tumor cells, then washed 
with an agent suspected of having tumoricidal ac- 
tivity, the animal laboratory has been used to 
continue studies into the mechanisms of tumor 
growth and dissemination. Proflavine hemisul- 
f ate in a 1 :500 or a 1 :1000 concentration has been 
shown to be extremely tumoricidal when tested 
against several tumor-host systems. While other 
drugs have decreased tumor growth, this agent has 
been consistently most active. Large animal tox- 
icity studies are now being completed in prepara- 
tion for a clinical trial. 

Attempts to alter growth of tumor by infecting 
a seeded wound with E. coli, Group A beta hemo- 
lytic streptococci, proteus vulgaris, and staphylo- 
coccus aureus revealed that only the streptococcal 
and coliform infections significantly decreased 
tumor growth. 



Cortisone appeared to increase the size of pul- 
monary metastases, the number of large lung tu- 
mors, and the growth of the primary T-241 Lewis 
sarcoma in C-57 BL/6JN mice. This effect was 
enhanced if cortisone was administered after 
tumor transplantation. When the primary tumors 
were amputated, this effect of cortisone was not 
demonstrated. Amputation of the primary tumors 
decreased the incidence of lung metastases. The 
earlier the amputation, the more pronounced was 
its effect. Cortisone did not alter the anatomical 
distribution of metastases. 

With the Millipore filter technique we were able 
to demonstrate tumor cells in the blood of off- 
spring of pregnant mice injected with S-91 mel- 
anoma. This passage across the placental barrier 
had been postulated but not demonstrated. 

Previous extensive laboratory investigations 
with S-91 melanoma and T-241 Lewis sarcoma 
have shown these tumors to metastasize only to 
the lungs. With individual housing, the life of 
the animal can be extended from 1 to 3 weeks. 
These mice will then frequently demonstrate 
metastases retroperitoneally to the liver and bowel. 

Malignant bronchial changes have not been ob- 
served in the 514 years during which a bronchial 
pouch in dogs was exposed to material thought to 
be carcinogenic. 

Laboratory techniques are being developed and 
standardized which may allow us to determine if 
autoimmunization of patients with their own tu- 
mors produces a specific immunological response 
or therapeutic effect. Skin tests, tannic acid 
treated hemagglutination tests, fluorescent anti- 
body techniques and cytotoxicity studies are being 
evaluated. Seven patients were treated with their 
own tumor in preliminary work. No significant 
response, either clinically or in the laboratory test- 
ing, was elicited. We would like to be able to de- 
tect and quantitate circulating antitumor anti- 
bodies and characterize these antibodies in terms 
of antigen specificity. 

Additional data have been accumulated refer- 
able to serum fractions which influence cell 
growth. Data accumulated to date indicates that 
serum from normal individuals contains two frac- 
tions, one capable of inhibiting cell growth and 
the other capable of stimulating cell growth of the 
regenerating rat liver. Concentrations of these 
fractions potentiate these effects. Serum from 
patients with cancer contains a stimulating frac- 



20 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tion but does not contain an inhibiting fraction in 
the same concentration as does serum from nor- 
mal individuals. This deficit appears to be of the 
magnitude of 75% or greater. The addition of 
the inhibiting fraction of normal serum to whole 
cancer serum or to the stimulating fraction of 
cancer serum produces inhibition of liver regenera- 
tion, indicating that the defect in cancer serum 
can be replaced. The periodic injection of the 
inhibiting substance into animals with a trans- 
planted tumor delays the time of appearance of 
the tumor and slows its growth rate. Mice in- 
jected with L-1210 leukemia and treated with in- 
hibiting substance live 10 to 12 days, while un- 
treated controls die at 7 days. 

With the acquisition of a full-time staff urol- 
ogist in the Surgery Branch, a number of projects 
have evolved which are based on problems of cur- 
rent interest in the field of urology. Of consider- 
able interest is the study of the levels of lactic acid 
dehydrogenase in the urine and the serum of 
patients in good health with disease. 

Renal vascular hypertension has been under 
study. The diagnostic work up includes the 1-131 
Hippuran renogram, bilateral renal function stud- 
ies performed by ureteral catheterization, as 
well as the usual clinical and chemical evaluations. 
Five patients have undergone corrective renal 
artery surgery for hypertension. 

Studies have been performed to evaluate the 
effect of Angiotensin II on renal function of sub- 
jects with normal blood pressure, hypertension, 
and unilateral renal artery disease. The normo- 
tensive and hypertensive kidneys respond to angio- 
tensin in different ways. Preliminary studies in 
the dog have shown a similar response. Sustained 
hypertension up to 6 months has been produced by 
partial ligation of segmental renal artery branches 
and then corrected by total artery branch ligation 
with relief of hypertension for up to 18 months. 

INTRAMURAL RESEARCH PROGRAM 

Introduction 

The translation of the data of biomedical re- 
search into benefits for the sick is often incomplete 
and always slow. These lags — conceptual, diag- 
nostic, therapeutic — are cumulative and even po- 
tentiating. The result is that in some diseases 
there is little resemblance between what is avail- 



able to the average patient and to the patient with 
the same disease at hospitals associated with 
active research groups. This lag can be excused 
in a number of ways, but underlying all of them 
is failure to accept responsibility for broad appli- 
cation of research information. The scientist and 
clinical investigators who establish a new finding 
are best able to recognize its implications, yet are 
in the poorest position to reduce it to general 
availability. The discoverers perhaps rightly feel 
their responsibilities end with publication. But 
the responsibility deficit is not in communication, 
for rapid communication of new findings already 
exists. The responsibility is that for action — to 
take new findings and to see that they reach the 
right patients through the complex social, eco- 
nomic, traditional, political and emotional milieu 
of our free society. 

During the past 2 years the physicians of the 
National Cancer Institute have been reviewing 
those research findings which should be applied 
broadly, and the means by which this might be 
done. The many new findings related to the acute 
leukemias led to a decision to form an Acute Leu- 
kemia Task Force. The membership* includes 
scientists and physicians who are making the new 
observations, clinicians with responsibilities for 
large numbers of leukemic patients and scientists 
engaged in drug development. A discussion with 
officials of a large industrial corporation which 
had used the task force mechanism effectively led 
to the conclusions that a task force should limit 
itself to one or at the most two objectives : set up 
criteria for their accomplishments; be dissolved 
when these end points were reached. The Acute 
Leukemia Task Force chose two objectives; (1) 
the extension of platelet and granulocyte replace- 
ment to many patients with acute leukemia; and 
(2) the establishment of accurate predictive ani- 
mal systems for selecting antileukemic drugs. 

The repair of platelet and granulocyte deficit in 
the depleted leukemic patient is part of routine 
care at National Cancer Institute. A number of 
patients who would have died from hemorrhage 
or infection are thereby saved and can often attain 



♦Membership : Dr. J. H. Burchenal, Dr. S. Farber, Dr. 
E. Frei, Dr. E. Freireich, Dr. J. Leiter, Dr. J. Louis, 
Dr. E. K. Marshall, Dr. T. McGinn, Dr. M. L. Murphy, 
Dr. H. E. Skipper, Dr. G. Taylor, Dr. P. Waalkes, Dr. 
R. Whittington, Dr. S. L. Rivers, Mr. W. Lourie, Dr. 
M. Sloan. 



NATIONAL CANCER INSTITUTE 



21 



additional drug induced remissions. If these 
techniques were available throughout the country, 
the average survival time of patients with acute 
leukemia would be lengthened, perhaps doubled. 
The major problem in extending this technique is 
that of platelet and granulocyte supply. In 1962 
6,000 units of blood were processed for platelets, 
as compared to 4,000 units required for the heart- 
lung apparatus and 4,000 for all other transfusions 
at National Institutes of Health. Additional limi- 
tations are placed on supply since platelets must 
be used within a few hours of collection. The 
Task Force has sponsored discussions among many 
interested parties and taken action to increase the 
supply of donors, improve harvesting by plas- 
mapheresis, develop a continuous flow centrifuge, 
and to study freezing and storage. It is even more 
difficult to obtain adequate numbers of granu- 
locytes and only chronic melocytic donors can 
supply the 100 billion cells needed per transfusion. 
To solve the supply problem, continuous plas- 
mapheresis of normal donors will probably be 
needed using a continuous flow centrifuge. 
Throughout all of these problems runs the need 
to expand the research and services of blood banks 
to meet the present and future clinical needs. 

The Task Force has made considerable progress 
with its second objective — the establishment of 
better predictive systems for selection of anti- 
leukemia drugs. Six drugs can induce complete 
remission in the acute leukemias — 6-mercapto- 
purine and methyl gas in acute myelocytic leuke- 
mia ; 6-mercaptopurine, prednisone, methotrexate, 
vincristine and cyclo-phosphamide in acute lym- 
phocytic leukemia. The L-1210 mouse leukemia 
successfully discriminates for four drugs but 
misses vincristine and prednisone. The Task 
Force with the cooperation of Cancer Chemothera- 
py National Service Center and other units of the 
Task Force has supported an examination of the 
six agents and a number of clinically negative 
compounds in a wide variety of animal leukemias 
and lymphomas. In this recent work Pi 534 leu- 
kemia seems to discriminate for vincristine. The 
goal is a battery of tumors sensitive to the six drugs 
and insensitive to clinically inactive agents. Ten 
additional compounds have passed the L1210 
screen and are in preclinical or clinical trial. Some 
have definite clinical activity, but their introduc- 
tion into broader clinical trial will depend on 
studies of degrees of effectiveness and toxicity. 



The Task Force has also had under study the 
problems of combination chemotherapy. If one 
considers the possibly analogous situation of tuber- 
culosis chemotherapy, it may be recalled that pul- 
monary tuberculosis could not be cured by either 
PAS or streptomycin. Cures were achieved when 
the drugs were given together. It is conceivable 
that a combination of two or more of the anti- 
leukemic drugs could bring about prolonged or 
permanent remission. The Task Force has devel- 
oped studies of combinations of the six drugs in 
the animal leukemias, in toxicity models and in 
patients. The ability to predict the toxicity or 
usefulness of drug combinations from animal sys- 
tems would be of great value. 

It had been planned to consider the viral etiology 
of the leukemias in the deliberations of the Acute 
Leukemia Task Force. When its goal was sharply 
focused, virus problems were removed from its 
scope. Research in the viral etiology of cancer 
had resulted in so many new findings it became ap- 
parent there was an opportunity to pursue more 
vigorously the viral etiology of some clinical 
cancers. It was decided to establish a second 
group — the Virus Task Force.* It has recently 
been formed and has as its central objective the 
rapid examination of the virus etiology of the 
leukemias. 

During the past year there have been several 
important observations on canced viruses by Na- 
tional Cancer Institute scientists. The murine 
models continue to be studied profitably, and there 
is gradually emerging a reconstruction of the 
morphologic events occurring between inoculation 
and appearance of leukemia. Of importance is the 
morphology of the murine virus in electron mi- 
crography. Using new negative staining tech- 
niques, the Moloney agent has been shown to have 
a tail and a hexagonal head not unlike the bacteri- 
ophages. It is apparently unlike the bacterio- 
phages in having a longer tail, lacking a complex 
injection apparatus, attacking mammalian cells 
and being composed of UNA. Similar morphol- 
ogy has been found for the Eauscher and Friend 
agents. This finding permits specific identifica- 
tion of these three agents. It has a further sig- 
nificance in that it will permit examination of 
mechanisms by which the virus affects the mam- 

*Members: Dr. W. R. Bryan, Dr. J. Grace, Dr. R. 
Huebner, Dr. F. Horsfall, Dr. P. Kotin, Dr. J. Melnick, 
Dr. R. Miller, Dr. R. Stevenson. 



22 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



malian cell. This is well understood for the bac- 
teriophages and bacteria, and similar studies can 
be done for the murine leukemia agents. A large 
effort is underway to uncover a viral agent in the 
acute leukemias and in stomach cancer. There are 
definite positive findings, but at the time of writing 
no clear cut proof of a clinical oncogenic agent 
exists. The importance of the use of research data 
in the control disease must not be allowed to ob- 
scure the realization that such data can come only 
from an organization with scientific excellence. 
The quality of the research during 25 years of 
National Cancer Institute is evidence enough that 
this has been achieved. More important is the 
resulting current staff of dedicated scientists and 
physicians who individually contribute so many 
new observations and collectively have such a 
broad and deep understanding of cancer. Both 
qualities are apparent in the accompanying sum- 
maries of the work of the past year by the Labora- 
tory and Branch Chiefs. Since it is not possible 
to review all the important new contributions, men- 
tion will be made of four areas of significance for 
the future. 

1. The need has been apparent for a tissue cul- 
ture medium which is chemically defined and free 
of serum and protein hydrolysates. Quantitative 
studies in cell nutrition cannot be done while 
serum or protein contribute unknown amounts of 
vitamins and amino acids. Neither can valid 
studies on virus growth in tissue culture be per- 
formed in the presence of added serum because 
of the possible presence of antibodies or viruses. 

Scientists of the Tissue Culture Section of the 
Laboratory of Biology are now able to carry 14 
cell strains in chemically defined media. With 
three strains of mouse fibroblasts, for the first time 
cells have been as hardy as those grown in the 
serum medium. 

2. Study of the relations of nuclei acids and 
proteins within the cell will not be precise until 
methods exist for the separation, purification and 
characterization of the various macromolecules. 
In the Biochemistry Laboratory emphasis has 
been placed on the perfection of techniques anal- 
ogous to those developed in the column separation 
of proteins. Considerable success has been 
achieved in purifying ribosomes and ribonucleo- 
proteins, oligonucleotides and lecithin and choline 
containing nucleotides. While advances are being 
made in purification of these macromolecules, 



much remains to be done. A somewhat different 
approach has been undertaken in the joint Oak 
Bidge-National Cancer Institute effort to develop 
high speed zonal ultracentrif uges. 

3. A knowledge of the interdependence of tumor 
and host as they make joint demands on nutri- 
tional resources would help to understand and per- 
haps indicate a way to control tumor growth. One 
of the scientists in the Biochemistry Laboratory 
has continued study of this problem in a system 
wherein a tumor inserted under a kidney or ovar- 
ian capsule of the rate develops a single arterial 
supply and venous drainage. He has shown that 
collagen of such a tumor is produced by cells of 
the host, not of the tumor. It has also been found 
that the interstitial fluid of this tumor has a con- 
stant composition in spite of tumor necrosis. It 
differs from composition of serum in that protein 
is 40% less, and contains no fibrinogen. Glucose is 
absent from the interstitial fluid unless there is 
marked hyperglycemia, but the lactic acid con- 
tent is twice that of serum. Cholesterol and lipid 
phosphorous are much lower than in serum. 
These discrepancies give a hint of marked dif- 
ferences in the metabolic patterns of this tumor 
and normal tissues. 

4. The successes achieved by the physical scien- 
tists in the use of computers have not been matched 
in biology. Perhaps this is because physical sys- 
tems can validly be studied in each of its parts, 
while living systems must always be studied 
whole. A more attractive possibility is that the 
physical scientist can move his problem directly 
to the computer while the biologist needs a middle 
man who understands biology and mathematics. 
Developments in the National Cancer Institute 
give some hope that the second explanation is cor- 
rect. For 2 years the Energy Metabolism Section 
of the Laboratory of Physiology has had a 1620 
computer. It is in use by a number of National 
Cancer Institute scientists with the help of mem- 
bers of this section. The following research stu- 
dies have been profitably programmed: 

(a) Analysis of arrangement of mononucleo- 
tides within polynucleotides derived from UNA. 

(b ) Effect of irradiation on production of sheep 
antihemolysins. 

(c) Retrieval and correlation of pathological 
diagnoses in patients. 

(d) Evaluation of dose-response phenomena in 
toxicity and antitumor effects of drugs. 



NATIONAL CANCER INSTITUTE 



23 



(e) Determination of the pattern of urinary 
excretion of nucleic acid metabolites by leukemia 
patients. 

(/) Another scientist has in collaboration with 
Dr. Mones Berman of National Institute of Aller- 
gy and Infectious Diseases and the Rand Corpora- 
tion programmed a Monte Carlo model for the 
effect of irradiation on the survival and growth 
of populations of cells. 

(g) Working with the Princeton computer 
group another scientist has programmed models 
for carcinogenesis due to ultraviolet light, X-rays 
and viruses. The success of these programs leads 
one to hope that the biomedical scientist will one 
day gain from the computer the same advantages 
enjoyed by the physical scientist. 

In considering the productivity of scientists and 
physicians of the National Cancer Institute, the 
question must be posed — "How is present excel- 
lence to be maintained in the future?" It is clear 
to all that this depends upon the recruitment of 
scientists and physicians of the highest quality 
and the creation of an atmosphere which will en- 
sure their retention and unlimited scientific devel- 
opment. The problems are known to all and need 
not be listed in their entirety. While salary levels 
and isolation from the academic life are impor- 
tant, the greatest problem is the growing convic- 
tion that the National Institutes of Health is of 
such size it can no longer respond to scientific need 
with the speed and flexibility essential to excel- 
lence. The intramural program of the National 
Cancer Institute is a fragment of a much larger 
National Institutes of Health complex, many of 
whose functions are not related to the needs of 
intramural research. If a new start were being 
made a National Cancer Institute, complete with- 
in itself, might be considered desirable. Yet much 
would be lost by such splendid efficiency — the con- 
tacts with many fine minds in the same and other 
disciplines, the great hospital which has been cre- 
ated with the seven institutes, and the attractive- 
ness of the National Institutes of Health to scien- 
tific visitors from the entire world. On the whole, 
the advantages of the larger complex to the 
National Cancer Institute outweigh the disadvan- 
tages. This does not relieve any of us from the 
duty of imaginative administration to create the 
ideal climate for the flowering of biomedical re- 
search. The quickest way to control disease is to 



make it easy for the best scientists and physicians 
to work at it. 

LABORATORY OF BIOCHEMISTRY 

Cytochemistry Section 

Drs. Woods and Burk have found that reduced 
diphosphopyricline nucleotide (DPNH) produced 
very marked inhibition of anaerobic glycolysis of 
intact ascites cells and certain other tissues. Ad- 
dition of excess oxidized diphosphopyridine nucle- 
otide (DPN), pyruvate or methylene blue, con- 
verting the added DPNH to DPN via reductases, 
could completely abolish this inhibition. The 
DPN/DPNH ratio is thus believed to be im- 
portant in tumor cell metabolism, and chemo- 
therapy and radiotherapy also. The ratio prob- 
ably dominates the glycolytic activity at the triose- 
phosphate-dehydrogenase step inside the cell. 
Many chemotherapeutically employed quinones 
may exert their effect via this DPN/DPNH ratio. 

Drs. Burk, Woods and Giger (NIH Post doc- 
torate Fellow) showed that methylglyoxal-bis- 
guanylhydrazone (MeGAGr) was a potent inhibi- 
tor of respiration in L-1210 mouse leukemic and 
other ascites tumor cells; much more so in 
MeGAG-sensitive than in MeGAG-insensitive 
lines. The inhibition was found to be localized at 
two DPN-dependent loci, namely malic and pyru- 
vic dehydrogenases, but at no other DPN- or TPN- 
mediated enzyme systems. Anaerobic glycolysis 
was not effected by MeGAG but aei*obic glycolysis 
was increased, as a result of the respiratory inhibi- 
tion, due to a Pasteur effect. 6 aminonicotamide 
(6-AN) both in vivo and in vitro enhanced the 
action of MeGAG on tumor cells and decreased its 
toxicity for the host. Detailed chemotherapeutic 
studies with 6-AN and MeGAG combinations are 
thus strongly indicated, particularly with mice. 

Dr. Woods and Mrs. Kolbye (guest worker) in 
collaboration with Dr. M. Landy are studjdng the 
possibility that cellular glycolytic responses of 
endotoxin represent a delay hypersensitivity re- 
action. This has led to observations that BCG- 
sensitized spleen and macrophages showed abnor- 
mally high aerobic glycolysis with altered response 
to injected endotoxin. Endotoxins appeared to 
increase nonspecific (phagocytic) and specific (an- 
tibody) resistance to infection. Low nonstressing 
injections of endotoxin could cause marked lower- 



24 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ing of glucose levels in normal and alloxan-dia- 
betic mice. 

Important differences between extravascular 
and intravascular leukocytes have been found by 
Dr. Evans (NIH Postdoctoral Fellow) in collabo- 
ration with Dr. Mueller (NIMH). They were 
able to induce myelopoiesis in guinea pigs by the 
intraperitoneal injection of sterile casein. Mar- 
row, exudate, and blood myeloid leukocytes all 
phagocytized particles such as polystyrene but 
only the first two took up fatty acid (albumin- 
bound palmitate). With marrow this uptake of 
fatty acid produced little respiratory stimulation, 
but with myeloid exudate cells there was a marked 
respiratory stimulation that was cyanide-insensi- 
tive. With blood myeloid cells (polymorphonuc- 
lear leukocytes (there was also marked, cyanide- 
insensitive respiration stimulation when polysty- 
rene particles were taken up. The glucose uptake 
of normal marrow cells was only one-third that of 
immature (casein-stimulated) marrow myeloid 
cells or of exudate myeloid cells. It appears that 
mature myeloid marrow cells have less glucose up- 
take and higher palmitate uptake than do the im- 
mature myeloid marrow cells. The respiration of 
exudate, and mature and immature myeloid cells 
was virtually the same. 

Further studies on the effects of vincaleukoblas- 
tine sulfate (VLB) have provided evidence that 
the alkaloid inhibits the Pasteur effect. This effect 
may well be involved in the causation of meta- 
phase arrest by the drug. Visible light can mark- 
edly increase the effects of added dyes on tumor 
cell metabolism, greatly increasing respiration but 
totally eliminating aerobic glycolysis (Hunter and 
Burk). 

Nucleic Acids Section 

The enzymes in normal and cancer tissues re- 
sponsible for the synthesis of the deoxynucleoside 
triphosphates needed for DNA synthesis are being 
studied by Dr. Kielley. She has found that the 
low thymidylate kinase activity of normal mouse 
liver as compared to hepatoma was apparently an 
artifact resulting from the fact that the enzyme 
was bound to particles in the liver. If the liver 
was frozen and thawed several times, the activity 
of liver extracts became the same as that of hepa- 
toma extract. She has also observed that normal 
liver could form deoxycytidine triphosphate much 
more efficiently than could the hepatoma. These 



findings have important implications with regard 
to the currently held concept that thymidylic 
kinase is a limiting factor in DNA synthesis. The 
nature of the thymidylate kinase bond to the par- 
ticles of normal liver is being studied. 

Dr. Rotherham has continued her studies of the 
deoxyribonucleosides of the tissues and urine of 
normal and tumor rats to examine the changes that 
occur in relation to the cancer process. Male rats 
have been found to excrete about five times as 
much deoxyribosidic material in the urine as do 
female rats. Castration of the males removed this 
difference. The deoxyribonucleosides present in 
the urine of male and female rats were the same 
with deoxycytidine accounting for approximately 
67% and 5-methyldeoxycytidine for 23%. No evi- 
dence could be obtained for the occurrence of the 
latter nucleoside in blood or tissues. 

Experiments by Dr. Ram Behki (Fellow of the 
Jane Coffin Childs Memorial Fund) and Dr. 
Schneider have shed new light on the intracellular 
locus of DNA synthesis. While the biochemical 
literature had indicated that DNA synthesis oc- 
curred in the soluble fraction of tissues, present 
experiments, however, show that the endogenous 
deoxyribosidic compounds of normal liver, regen- 
erating liver and hepatoma were present in the 
nucleus provided that the nucleus was isolated in 
such a way as to prevent redistribution, i.e., the 
use of lyophilized tissues and of dry organic sol- 
vents of graded densities for the isolation of the 
nuclei. When aqueous solutions were used, these 
compounds were recovered in large part in the sol- 
uble fraction and considerable degradation of 
nucleotides occurred. A study of DNA polym- 
erase led to similar findings, i.e., the enzyme was 
associated with nuclei when these were isolated by 
the nonaqueous method. It appears, therefore, 
that the precursors of DNA as well as the DNA 
polymerase are normally found in the nucleus but 
are lost when nuclei are isolated in aqueous media. 

Studies by Dr. Schneider on the enzymatic syn- 
thesis of deoxycytidine diphosphate choline and 
its utilization for lecithin formation were contin- 
ued in an effort to discover the reason for the oc- 
currence of this nucleotide in the Novikoff hepa- 
toma. The activity of the enzyme responsible for 
the synthesis of dCDP-choline in extracts of both 
normal liver and the Novikoff hepatoma was found 
to increase several-fold when tissue preparations 
were aged at 0° for several days, incubated at 37° 



NATIONAL CANCER INSTITUTE 



25 



for three hours, dialyzed or passed through 
Dowex-50-Na + columns. The enzymatic synthesis 
of lecithin was stimulated 5-fold by adding an 
emulsion of diglyceride to the reaction mixture. 
This stimulation occurred in both liver and hep- 
atoma although the ability of the latter to form 
lecithin was considerably less than that of the 
liver. However, CDP-choline was much more 
active in lecithin formation than was dCDP- 
choline in both liver and hepatoma and in the pres- 
ence or absence of added diglyceride. These ex- 
periments provide further support for the pre- 
vious conclusion that the occurrence of dCDP- 
choline in the hepatoma is related to the limited 
ability of this tissue to form phospholipids but 
leave open the possibility that this compound may 
be used in DNA synthesis. 

As part of a continuing study of structure and 
sequence of nucleic acids, work on the nucleotide 
distribution of ribonucleic acids, on the develop- 
ment of enzymatic methods for the cleavage of 
nucleic acids at specific points, on the development 
of fractionation methods, and on the preparation 
and characterization of oligonucleotide sequences 
has been continued by Drs. Rushizky and Sober 
with the collaboration of Dr. E. Bertos (NSF 
Postdoctorate Fellow) and Dr. Edith Townsend 
(Visiting Fellow). They have been able to show 
with Dr. C. A. Knight of the Virus Laboratory, 
University of California, that the RNA obtained 
from various strains of tobacco mosaic virus could 
be differentiated on the basis of the nucleotide 
products produced after hydrolysis by pancreatic 
RNase and RNase T a . Nucleases from Takadias- 
tase and from B. sub tills have been purified and 
characterized with respect to their hydrolytic spec- 
ificity. RNase Ti, obtained from Takadiastase, 
showed strict specificity for the hydrolysis of the 
5'-P0 4 linkage distal to the guanylic acid residue 
leaving a terminal 3'-P0 4 guanylic acid. How- 
ever, RNase T 2 , also from Takadiastase, did not 
show a strict hydrolytic specificity but rather only 
a preference for adenylic residues. Complete di- 
gestion by this enzyme resulted in complete reduc- 
tion of the nucleotide chain to mononucleotides. 
This enzyme has become extremely useful for the 
determination of nucleotide base composition. The 
RNase from B. subtilis has been purified exten- 
sively. It has a low molecular weight but only 
shows partial purine specificity, favoring guanylic 
residues over adenylic residues but ultimately re- 



ducing the whole nucleic acid chain to the di- and 
mononucleotide stage. In the course of the above 
studies, most of the di-, and tri-, and tetranucleo- 
tide sequences of RNA have been prepared and 
characterized by a combination of chromatogra- 
phic and electrophoretic methods. They are being 
used as model and standard substances for meas- 
urements of a number of physicochemical param- 
eters and enzyme specificity. Studies of reactiva- 
tion of the immune response of immunologically 
suppressed animals by DNA and its degradation 
products have been initiated with Dr. M. Feldman, 
Laboratory of Experimental Biology, Weizmann 
Institute of Science, Rehovoth, Israel. 

Nutrition and Carcinogenesis Section 

The development by Dr. Morris of a large num- 
ber of transplantable rat hepatomas showing much 
similarity to normal rat liver has led to the con- 
cept of a "minimal deviation tumor." The great 
interest in these biochemically interesting tumors 
has led to a large number of cooperative investi- 
gations of their enzymatic patterns and their 
metabolism. Much detailed information of indi- 
vidual systems has been obtained but, in general, 
these tumors exhibit enzymatic distribution pat- 
terns which are more like those of normal liver 
than the rapidly growing hepatoma. One of the 
most important observations to date is that no two 
of the minimal deviation tumors are alike. The 
enzyme patterns themselves and responses to 
physiologic stimuli show striking individual 
differences. 

Further studies of experimental gastric cancer 
in the rat have revealed for the first time that this 
cancer can be induced by N,N'-2,7-fluorenylene- 
bisacetamide (2,7-FAA) injected into the animal 
at a site distant from the site of the cancer. 
Studies are underway on inhibitors and promoters 
of 2-fluorenylacetamide carcinogenesis. Although 
these are long term experiments, and all details 
have not yet been completed, it appears that blad- 
der tumors can be produced in Fischer strain rats 
without the addition of tryptophan. Jaundiced 
rats ingesting the carcinogen developed as many 
liver tumors as non jaundiced animals of the same 
genetic background. 

Biochemical studies in experimental cancer by 
Drs. Dyer and Morris have shown that the metab- 
olism of 2-FAA by the Rhesus monkey resembles 
that of the guinea pig, a species resistant to indue- 



26 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tion of cancer by this agent. However, the monkey 
unlike the guinea pig does not excrete N-OH-2- 
FAA glucuronide (a potent carcinogenic metabo- 
lite in the rat) after 2-FAA ingestion. 2-Fluo- 
renyldiacetamide, 2-fluorenylbenzoylamide and 
2-fluorenylphthalamic acid may owe their carcino- 
genic activity to the production of 2-FAA in vwo. 
Differences in carcinogenic activity may be due 
to the rate of absorption and to the amount of un- 
metabolized material excreted by the large in- 
testine. 2,7-Fluorenylenebisacetamide, a potent 
carcinogen for the rat and the dog, however, does 
not yield 2-FAA. Hydroxylated metabolites are 
excreted after feeding 2,7-FAA to both species. 
The dog, unlike the rat, excretes a large amount of 
unstable free amino metabolites of administered 
2-FAA and of 2,7-FAA. Tryptophan pyrrolase 
cannot be detected in some minimal deviation 
hepatomas but is present in others in low concen- 
trations compared with liver. In tumor lines 
where activity is detectable, that activity is in- 
creased by intraperitoneally-administered L-tryp- 
tophan. 

In attempts to study the effect of natural and 
purified diets on tumor induction and growth, Mr. 
Otey and Dr. Birnbaum have initiated studies in 
rats in which dimethylbenzanthracene is fed by 
stomach tube. Preliminary experiments have in- 
dicated that tumor induction was relatively slow 
on both diets with a slight variation in tumor inci- 
dence tentatively ascribed to the difference in food 
intake. In attempts to obtain a 100% incidence 
of tumors all locally available strains were tested 
and it was confirmed that the Sprague-Dawley 
strain Avas the most susceptible. Since in the early 
experiments there appeared to be some relation- 
ship between the age of weanling and the develop- 
ment of mammary tumors, current experiments 
are studying the effect of adding progesterone and 
anhydroxyprogesterone to the synthetic liquid 
diets. 

Preliminary results indicate an earlier appear- 
ance of the first palpable tumor. 

It has been shown by Dr. S. Birnbaum and Mr. 
Otey in their studies on the nature of renal amino- 
peptidase, that, like many other intracellular pep- 
tidases, the renal enzyme was activated by but not 
necessarily dependent on metal ions. Further 
work has been discontinued clue to Dr. Birnbaum's 
transfer to the Division of Research Grants. 



Prolein Chemistry Section 

A comprehensive investigation of the "Bence- 
Jones" proteins produced by mouse plasma cell 
tumors has been undertaken by Drs. E. L. Kuff, 
M. Potter, R. Mclntyre, and W. Dreyer 
(NIAMD). This has revealed that the Bence- 
Jones protein elaborated by tumor RPC 20 has a 
molecular weight of 24,000, that it contains one 
reactive sulfhydryl group per molecule, and that 
it is different from every other murine Bence- 
Jones protein thus far studied in its immunologi- 
cal properties and in the peptide "fingerprint" 
produced by tryptic hydrolysis. It forms a disul- 
fide dimer and also a hybrid molecule with serum 
albumin. It appears in the urine partly in the 
form of dimer and partly in combination with 
oligopeptides linked apparently through the sulph- 
hyclryl group, since the latter is no longer free. 
The murine Bence-Jones proteins can be divided 
into two classes on the basis of ultracentrifugal 
analysis : one with sedimentation rates near 2.8S 
and the other with rates between 3.3 and 3.9S, 
with both classes heterogeneous by starch gel elec- 
trophoresis. The situation is thus analogous to 
that observed in human multiple myeloma. 

Dr. W. Hymer (NIH Post Doctorate Fellow) 
and Dr. E. L. Kuff are developing methods for 
the isolation of nuclei from plasma cell tumors and 
have found that these exceptionally fragile com- 
ponents of the cell are stabilized by treatment with 
Triton X-100, a nonionic detergent. Similarly, 
very clean nuclei have been obtained from normal 
liver and kidney by dispersing other particulate 
cell components with this detergent. The liver 
nuclei retained their DPN-synthesizing capacity 
after treatment. The use of Triton X-100 has 
also made possible the isolation of tubular casts 
from the kidneys of tumor-bearing animals, and 
their composition is now under study. 

Drs. E. A. Peterson and E. L. Kuff, in collab- 
oration with Dr. R. W. Hendler (NHI), have 
found that ribosomes isolated by the column meth- 
od from normal rat liver labeled in vivo with ra- 
dioactive amino acids were markedly less radioac- 
tive than ribosomes isolated by conventional ul- 
tracentrifugal procedures. However, the micro- 
somal lipoprotein fraction washed off the column 
by Triton X-100, a nonionic detergent, was ex- 
tremely radioactive. The results suggest that the 



NATIONAL CANCER INSTITUTE 



27 



radioactivity associated with liver ribosomes iso- 
lated by the ultraeentrifugal method may be due 
to a small amount of active, lipid-containing sub- 
stance contaminating them, as has been recently 
found to be true of E. coll ribosomes. This active 
substance may have emerged from the column in 
the highly radioactive Triton X-100 eluate, 
which is known to contain newly synthesized pro- 
tein. 

Isolation and fractionation of the ribosomes of 
mouse plasma cell tumor RPC 20 by the column 
procedure previously described (1960) yielded a 
pattern similar to that obtained from rat liver, 
but the two major peaks were eluted at signifi- 
cantly lower salt concentrations than the corre- 
sponding liver ribosomes. Chromatography of 
microsomal RNA on ECTHAM-cellulose has been 
complicated by the large size of the molecules, 
their high density of negative charge, and their 
lack of a fixed configuration. It was necessary to 
use relatively high initial salt concentrations to 
minimize chromatographic anomalies that arise 
from the extended configurations assumed by 
RNA molecules at low salt concentrations because 
of repulsion between neighboring charges. It is 
of interest that the major portion of the RNA 
emerged from the column in the same region as the 
intact ribosomes when chromatographic condi- 
tions were the same. This suggests that the pro- 
tein of the ribosome is recessed within an RNA 
structure that dominates the surface of the 
particles. 

Dr. R. W. Hartley, Jr., in continuing his studies 
on the heterogeneity of crystalline bovine plasma 
albumin has found it possible to separate gram 
quantities of the components having a high con- 
tent of sulfhydryl groups from those having a low 
one by scaling up the sectioned column procedure 
previously reported. Chromatography on Sepha- 
dex G-200 was found to fractionate albumin 
purely on the basis of molecular weight, and sepa- 
ration of monomer and dimer from each other and 
from higher polymers was accomplished in a sin- 
gle pass. Equilibrium ultraeentrifugation of the 
dimer fraction provided the first unequivocal 
demonstration that this commonly observed al- 
bium component (S 20 of 6) does have a molecular 
weight just twice that of albium monomer. 

Dr. Hartley has collaborated with Drs. G. TV. 
Rushizky and H. A. Sober in the purification and 



characterization of an extracellular ribonuclease 
from Bacillus subtilis. Phenol extraction fol- 
lowed by a series of precipitation and chromato- 
graphic procedures yielded a product pure enough 
for molecular weight determinations which have 
indicated a value in the 10,000-12,000 range. This 
was supported by its behavior in chromatography 
on Sephadex G-75. The relatively small size, sta- 
bility, reported absence of disulfide bridges, and 
easily measured activity of this enzyme recom- 
mend it for a study of the relation between enzyme 
structure and function. 

Mrs. Mary TVyckoff and Dr. Peterson made a 
detailed study of the effects of temperature (0 to 
60°) on the ionization of the acidic and basic 
groups of several cellulosic ion exchanges and 
found that both types became progressively weaker 
as the temperature increased. Since similar ef- 
fects were observed when simple soluble com- 
pounds containing the same groups were titrated 
in free solution, the phenomenon appeared to be a 
result of the effect of temperature on the dielectric 
constant of water rather than on the structure of 
the adsorbent, itself. Because of an inflection in 
the temperature vs. ionization curves of the 
amines, the ionization of these (including "tris" 
buffer and DEAE-cellulose) was essentially the 
same at 5° and 25°, accounting for the nearly 
identical patterns obtained when serum proteins 
were chromatographed at these two temperatures. 
Albumin, however, was significantly displaced in 
the direction of tighter binding at the higher tem- 
perature, apparently because of some unusual 
property of its own. 

Difficulties previously interfering with the iso- 
lation and characterization of the metal proteins 
of plasma have been overcome. Drs. S. R. Him- 
melhoch (Research Associate), H. A. Sober and 
E. A. Peterson, in collaboration with Dr. Bert 
Vallee of Harvard University have perfected 
procedures for the routine removal of contaminat- 
ing trace metals from buffers, adsorbents, and 
equipment, and preliminary experiments have 
shown several metals (Ca, Mg, Ba, Zn, Cr, and 
Fe) to be present in specific fractions of dialyzed 
plasma. Larger scale experiments are expected 
to permit the detection of additional trace metals. 
It is believed that this work will ultimately aid in 
the interpretation of metal fluctuations in the 
plasma proteins in disease by revealing the changes 



28 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



in individual metalloprotein components instead 
of net changes in the total amount of a given metal. 

Drs. A. Yaron (Visiting Fellow) and H. A. 
Sober have undertaken the preparation and exami- 
nation of a variety of single and mixed amino acid 
polymers of known chain length, composition, and 
sequence for use as model substances in studies of 
the relationships of the structure of proteins to 
their biological activity and their chromatographic 
behavior. Homologous poly-alpha-amino acid 
mixtures having different average chain lengths 
(prepared by Drs. A. Berger, E. Katchalski, and 
M. Sela in the Department of Biophysics, Weiz- 
mann Institute of Science, Eehovoth, Israel) have 
been chromatographed on CM-cellulose and 
DEAE-cellulose to resolve the individual com- 
ponents. Besolution has been studied as a func- 
tion of temperature, pH, buffer, and load. Ade- 
quate resolution of the lower homologs of poly- 
lysine was obtained with a one gram load, and a 
scaling up of several fold is contemplated. Elut- 
ing systems have been devised that do not interfere 
with examination of the effluent at 220 micro- 
curies, and techniques have been developed for the 
recovery of the polyamino acid fractions from 
the effluent by precipitation. 

Dr. T. T. Otani has tested the N-chloroacetyl 
derivatives of a number of Beta hydroxyamino 
acids for growth inhibitory effects in three micro- 
bial systems as a preliminary screening for possible 
antitumor activity, N-Chloroacetyl-Beta-hydrox- 
yleucine and N-chloroacetyl-Beta-hyclroxynorleu- 
cine were the best inhibitors of microbial growth. 
When these two compounds were tested in collab- 
oration with Mr. John Venditti for antitumor 
activity in leukemic mice (L-1210) significant de- 
crease in tumor nodule size was noted, although 
there was no increase in survival time. One of the 
10 mice receiving N-chloroacetyl-Beta-hydroxy- 
norleucine at a level of 500 mg/kg/day shoAved 
complete regression of the tumor nodule and 4 of 
10 mice showed significant decrease in tumor size 
after 1 dose of the compound. While no significant 
loss of weight was observed in the animals before 
death, there were more deaths among the treated 
animals than among the controls on the third day 
of treatment. The possibility that this was the 
result of an acidotic condition deriving from the 
action of tissue acylases on the chloroacetyl deriv- 
atives will be taken into account in future tests. 



Tumor-Host Relations Section 

Increased efforts are being made to investigate 
the relationships between the tumor cells and the 
various tissues of the host. This has required the 
development of new in vivo and in vitro techniques 
and has resulted in an increased amount of collab- 
oration and consultation with colleagues in the 
Laboratories of Immunology, Virology and 
Biology. 

In studies which are exploiting the "tissue iso- 
lated" tumor technique of Dr. Gullino the devel- 
opment of connective tissue during the growth of 
the tumor has been investigated. It has been 
found that although the host fibroblast produced 
the collagen in the tumor, the concentration of 
collagen was controlled by the tumor type. In 
addition, it has been demonstrated that the col- 
lagen formed in the tumor was predominantly of 
the adult type. By the development of a small 
diffusion chamber which can be implanted in the 
tumor and connected to the exterior of the animal 
through a very fine capillary, studies of the inter- 
stitial tissue of the tumor during tumor growth 
have become possible. The interstitial fluid was 
characterized by a constant composition despite 
the presence of necrosis, a protein concentration of 
less than 40% that of plasma, an amino acid level 
equal to or higher than that of serum, the absence 
of fibrinogen, a negligible content of free glucose, 
a lactic acid concentration of 2- to 3-fold higher 
than serum, and a cholesterol and lipid phos- 
phorus content much lower than that of aortic 
serum. 

Dr. Shelton, with the aid of intraperitoneally 
implanted diffusion chambers, has studied the in- 
teraction between normal and tumor cells. She 
has found that small numbers of lymphocytic 
tumor cells markedly inhibited the collagen for- 
mation of norma peritoneal cells, but that hepa- 
toma cells had a much less striking effect on con- 
nective tissue fibroblasts. She has been able to 
show replication of the Moloney leukemia virus, 
after 38 days but not at 9 days, when grown to- 
gether in the chamber with new-born BALB/c 
mouse thymus. In collaboration with Dr. Vir- 
ginia Evans, she has demonstrated that fibroblasts 
were transformed into malignant cells in the milli- 
pore diffusion chamber. This finding provides an- 
other example of the malignant transformation in 



NATIONAL CANCER INSTITUTE 



29 



an essentially in vivo environment quite different 
from that of tissue culture. 

Dr. Eechcigl's studies on the multiple factors 
effecting catalase level of tissues has done much to 
clarify some of the relationships between the cata- 
lase content of liver in a tumor-bearing host with 
the growth of a neoplasm. Nutritional studies 
have indicated that amino acid deficiency may be 
one of the mechanisms by which the catalase re- 
duction in liver of the tumor-bearing rat is medi- 
ated. It was shown that the stress of a protein- 
free diet produced a characteristic lowering of 
liver catalase in both normal and tumor-bearing 
animals. However, in hepatoma 5123, a minimal 
deviation tumor, the catalase level was not altered 
under these same protein-free conditions. Sex, 
age, and location of tumor did not affect the tumor 
catalase level. In some tumors, namely, the trans- 
planted hepatoma 5123, the ethionine-induced 
hepatoma of rats and the spontaneous hepatomas 
of C3H mice, high catalase activity was found in 
the tumor tissue even though a definite depression 
in catalase content was observed in the liver and 
kidneys of these tumor-bearing animals. Trans- 
plantation of a single, primary ethionine-induced 
hepatoma into OM/N rats gave rise to two lines of 
hepatomas, one possessing extremely high catalase 
activity, sometimes exceeding that of the normal 
liver, and the second with a low catalase activity. 
No other significant chemical or enzymatic differ- 
ences have been obtained in these 2 hepatoma lines 
which have been carried through 20 transplant 
genei^ations to date. 

In preliminary studies with Dr. Heston it was 
found that most strains of the C57 Bl mice had 
half the normal liver catalase concentration except 
for the C57 Bl substrains, C57 Bl/He and C57 
Bl/An, which had values similar to that found for 
the liver of other mouse strains. These studies 
indicate the genetic control of the catalase content 
of liver. In collaboration with Dr. Wollman it 
was demonstrated that the removal of the pituitary 
gland does not prevent or overcome the charac- 
teristic catalase depression in the tissues of the 
tumor-bearing host, previously reported by Utsugi. 
The catalase depression was present in the tumor- 
bearing rats, whether hypothosectomy was per- 
formed before or after tumor implantation. Pre- 
liminary studies with Dr. Warren Evans (NIH 
Postdoctorate Fellow) have shown that when the 
catalase of leukocytes was inhibited with amino- 



triazole the respiration and glucose uptake was not 
altered. 

Development by Dr. Greenfield of methods for 
the in vitro maintenance of hepatic cells in func- 
tional state progresses nicely. A high oxygen re- 
quirement of liver cell suspensions has been estab- 
lished even at 0° C. in polyvinylpyrrolidone- 
sucrose mixtures. When maintained in growth 
media under 95% 2 — 5% C0 2 , the cells reaggre- 
gated at the miniscus. Initial studies using 95% 
2 — 2% C0 2 at 2 atmospheres appears promising 
since under these conditions the cells formed uni- 
form round aggregates on the bottom of the flask. 
Cells in the aggregates maintained respiration and 
gave other evidences of viability over a period of 
48 hours. 

Additional effects of the Lactic Dehydrogenase 
Agent have been demonstrated. Infection by this 
viral agent has been found to produce an eleva- 
tion in both normal and tumor-bearing mice of a 
number of plasma enzymes besides lactic dehydro- 
genase, namely : glutamic-oxalic transaminase, iso- 
citric dehydrogenase, malic dehydrogenase, and 
phosphohexose-isomerase. As has been reported 
previously for lactic dehydrogenase, the elevation 
of these enzymes is much more striking in the 
tumor-bearing animal than in the normal animal. 
The plasma aldolase levels were, however, not 
affected by the virus in either the normal or tumor- 
bearing animal. 

Studies of immune phenomena in mouse histo- 
compatibility systems have been initiated by Drs. 
Mishell (Research Associate) and Greenfield. 
Since analysis of histocompatibility loci has been 
handicapped by the lack of suitable in vitro tests, 
development of methods for detecting in vitro anti- 
body reactions which reflect histocompatibility 
loci are underway. Studies of the mouse leuko- 
cyte agglutination system are being extended to 
determine which histocompatibility loci determine 
these antigens. In early results it has been found 
that the BALB/c mice have been found to pro- 
duce satisfactory leukocyte agglutinins when hy- 
perimmunized with DBA/2 normal tissues. 

LABORATORY OF BIOLOGY 

The Laboratory of Biology continues to main- 
tain a broad biological approach to the cancer 
problem. Emphasis is on etiology. This year 
the Tissue Culture Section was added to the pro- 
grams of the Carcinogenesis Section and the Gen- 



30 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



eral Biology Section. With the single cloning 
techniques and the chemically defined media in 
which cells can now be grown directly from the 
animal, and with the inbred mouse to which the 
tissue can be returned for testing for malignancy, 
this section is in a good position to study malig- 
nant transformation. 

Carcinogenesis 

Dr. Deringer has continued her studies of the 
multipotential carcinogenic action of urethan 
using hairless strain Hr mice as test animals for 
skin carcinogenesis and strain DBA/2eB mice as 
test animals for induction of leukemia. Urethan 
painted on the skin of the hairless mice signifi- 
cantly increased the occurrence of epidermoid car- 
cinomas and decreased the latent period of both 
epidermoid carcinomas and papillomas. Urethan 
painted on the skin of DBA/2eB mice increased 
the occurrence of reticulum cell neoplasms and 
they arose earlier. Most of these were lympho- 
cytic neoplasms, whereas most of those in the un- 
treated controls were reticulum cell neoplasms of 
types A and B. The painted Hr mice also de- 
veloped lung tumors and both the Hr and 
DB A/2eB mice developed multiple hepatomas and 
hemangioendotheliomas of the liver. Hemangio- 
endotheliomas of the Hr mice were transplatable. 

Using a genetically more resistant strain C3H/ 
Lw, Dr. Law found that the urethan administered 
to neonatal mice was ineffective in inducing leu- 
kemia and even inhibited leukemia in C3Hf/Lw 
mice carrying the Moloney leukemia virus. Mul- 
tiple hepatomas were found in the treated mice. 

Dr. Law has found that thymectomy within the 
first 24 hours following birth and at 1 month pre- 
vents lymphocytic neoplasma in his three high 
leukemic C3H sublines. However, in those ani- 
mals in which thymic remnants remain granulo- 
cytic leukemias were found relatively late in life. 
Splenectomy at birth and at 1 month has been in- 
effective in influencing occurrence of leukemia in 
the three high leukemic C3H sublines which raises 
the question of the significance of those histopath- 
ogenic changes described in the spleen preceding 
the appearance of leukemia. Isologous thymic 
grafts introduced under the kidney capsule fol- 
lowing standard, fractionated, X-irradiation did 
not develop into lymphosarcomas nor induce leu- 
kemia in C57BL/Ka recipient mice, but such 
such grafts given subcutaneously became leukemic. 



Dr. Potter has continued the study of induction 
of plasma cell tumors with mineral oil (Bayol F) . 
Such tumors can be induced in BALB/cAn mice 
and in Fj hybrids resulting from outcrossing to 
the closely related strain A, but not in a number 
of other strains including A or in other Fi hy- 
brids. Plasma cell tumors have also been induced 
in BALB/c mice with Drakeol-6VR which has 
been used clinically because it was thought not to 
be carcinogenic. Subcutaneous sarcomas have 
also been induced with incomplete Freund's ad- 
juvants and to a lesser degree with Bayol F. 

In studying the mammary tumor inducing ef- 
fects of added pituitary glands, Heston and co- 
workers were able to induce mammary tumors in 
genetically resistance C57BL virgin females with 
the implantation of five extra pituitary glands 
under the capsule of the kidney. None were in- 
duced with one extra pituitary gland. This il- 
lustrates the relative importance of hormonal fac- 
tors and the mammary tumor virus. These C57BL 
females did not have the virus and it is difficult to 
induce mammary tumors in them with the virus, 
especially when they are kept as virgins. 

Immunology 

Because of Miller's report of a wasting illness 
of mice thymectomized at birth, Dr. Mclntire has 
been interested in this procedure to study the role 
of the thymus in leukemogenesis and in immunol- 
ogic responsiveness. His technique of removing 
the thymus within the first 24 hours of birth re- 
sulted in a low mortality (10-20%) compared 
with (75-100%) reported by others, but later at 
autopsy there was no evidence of residual thymic 
tissue. Many of the animals became weak with 
weight loss and ruffled hair and some had diarrhea, 
but they had normal or only slightly decreased 
lymphocytic tissue. Only 1/20 thymectomized 
mice had a delayed rejection of homologous skin 
which did not differ at the H-2 locus. Natural 
antibody titers to gram negative bacilli of these 
mice were no different from sham thymectomized 
litter mates. Failure to confirm the reports of 
Miller suggests that there may be other factors 
in the illness and death following neonatal thy- 
mectomy, the most likely being viral infection. 

Miss Uphoff has continued her program on im- 
munologic responses of irradiated mice and pro- 
tection with bone marrow transplantation. One 
observation bearing directly on the cancer prob- 



NATIONAL CANCER INSTITUTE 



:u 



lem was the high incidence of leukemia in AKR 
mice that had received 700r X-irradiation and 
protected with bone marrow from low-leukemic 
strains C3Hf, CBA, ST, and the high-leukemic 
strain C58. Transplantation proved most of these 
neoplasms to be of donor tissue origin. Similar 
results were obtained from F a hybrids (ARK x a 
low-leukemic strain). Low-leukemic strains pro- 
tected with AKR marrow had a low leukemia 
incidence and all of these neoplasms were of AKR 
tissue origin. 

Virology 

Work in this area is concerned with the biology 
of tumor viruses in experimental animals. 

Two mouse leukemia inducing viruses have been 
discovered by Law and coworkers during the past 
year. P-LLV was isolated from a 5 month old 
C3Hf/Bi mouse. BALB/c, C3Hf/Bi, and 
C3Hf/Lw mice inoculated during the neonatal 
period with the filtered agent developed lympho- 
cytic neoplasms at 3 months, but DBA/2 and 
C57BL/Ka mice did not. Passage of P-LLV 
through the mother's milk was observed (it may 
later be shown that this is the same as the Moloney 
virus). S-180LV was isolated from Sarcoma 
180. This virus induces leukemia, principally 
granulyocytic in C3H mice and lymphocytic in 
BALB/c. (This apparently is not the same as 
any other known leukemia virus.) 

In his continuation study of the natural transfer 
of mouse lekemia viruses, Law has found that they 
are transferred readily through the milk but 
scarcely or not at all prenatally from the mother 
or from the father. The extrachromosonal pat- 
tern for congential transmission of these viruses 
stands in sharp contrast to the chromosomal pat- 
tern of transfer potentialities to develop leukemia 
in the high leukemia, strains such as AKR, C58, F, 
and C3Hf/Fg and raises the important question 
of whether viruses are involved in the etiology of 
the disease in high leukemia lines. 

Dr. Andervont has shown that the mammary 
tumor virus can disappear from certain sublines 
of strain RIII mice. These lines are remaining 
low tumor lines. New evidence this year indicates 
that this disappearance is not limited to the RIII 
agent in RIII mice but that the potent C3H agent 
introduced into RIII can also disappear from 
certain lines. 



Further studies by Dr. Andervont on transmis- 
sion of the mammary tumor agent by the male 
indicate that the foetus can be infected directly in 
utero. The agent does not pass through the wall 
of the uterus to infect the mother. 

Dr. Barrett has been studying the long latent 
period of the mammary tumor virus. By injecting 
agent-free but susceptible mice of different ages, 
(newborn, 1 month, and 10 months) he showed 
that the latent period of the tumors was about the 
same in all groups. They became less susceptible 
with advancing age. These results suggest that 
the "latency" represents a maturation of the host- 
virus relationship and does not represent aging in 
the host tissue. 

To test the concept held by some investigators 
that the low mammary tumor incidence in C3HfB 
mice is due not to their being free of the agent 
but to their having obtained a duplicating inhibi- 
tor from the foster strain C57BL, Heston and 
Vlahakis have started a strain C3Hf C by fostering 
C3H on BALB/c. BALB/c lacks the agent but 
is susceptible to it and theoretically then would 
not have the inhibitor. According to this concept 
the C3HfC, free of inhibitor, should have a high 
tumor incidence. This has not been observed. We 
still believe that these agent-free lines have a low 
incidence of tumors because they are agent-free. 

Doctors Sanford and Law have been studying 
the effects of polyoma virus on cell transforma- 
tions in vitro. All but one strain tested have shown 
the malignant change and in certain cases the virus 
treated cells showed this change earlier than the 
untreated controls. Antigenic alteration has been 
obtained. Experiments are still in progress to 
determine whether the malignant and anti- 
genic changes are associated or are independent 
phenomena. 

Cell Physiology and Nutrition 

The Tissue Culture Section made an important 
contribution this year in getting freshly explanted 
mammalian tissue cells to grow directly in protein- 
free chemically defined culture media. Three 
strains of mouse cells started in this manner have 
been grown continuously. This is the first time 
cells have been grown in chemically defined media 
without first undergoing a period of adaptation in 
media to which serum had been added. It will rep- 
resent a great advancement for the study of cell 



32 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



nutrition, for attacking the problem of the malig- 
nant change, and for virus work using tissue cul- 
ture. It ranks in importance with the single cell 
cloning and the chemically defined media. This 
contribution can be contributed directly to the 
work of Dr. Evans and Mr. Bryant, but of course 
would not have been possible without the work of 
the whole section in methodology and cell nutri- 
tion. 

Further advance has been made in growing a 
variety of cells in chemically defined media after 
the period of adaptation. Fourteen established 
cell strains from man, mouse, monkey, and ham- 
ster are now growing in chemically defined media. 
This gives rise to the hope that any cell strain 
that can be grown in undefined media can ulti- 
mately be adapted to chemically defined media. 

In their studies on cell nutrition, Dr. Sanford 
and associates have shown that serum protein when 
added to otherwise chemically defined media pre- 
vented several vitamin and amino acid deficiencies. 
It appears that the serum protein contributes ap- 
preciable amounts of vitamins, amino acids, and 
other components of the cells. These results raise 
serious doubts that any accurate quantitative stud- 
ies on the nutrition of cells can be carried out on 
a media containing serum protein supplement. 

In a protein free media biotin was identified for 
the first time as a vitamin required for survival of 
a mammalian cell strain in culture, and vitamin 
B i2 was shown to be necessary for maximal pro- 
liferation of certain but not all cell types. With 
the strain of mouse fibroblasts the requirement for 
biotin could be demonstrated only when the media 
lacked the purine nucleic acid derivatives and 10 
of the 18 vitamins. Vitamin B 12 was demonstrated 
to prolong the survival of cells when the media 
lacked folic acid. Results of studies of effects of 
16 other vitamins have led to formulation of sim- 
pler media than had formerly been practicable. 

In chemically defined protein free media, galac- 
tose was found to be utilized in place of glucose. 
The study is being pursued in order to correlate 
the activity of enzymes for galactose utilization 
in 929-L cells with growth responses to galactose. 
Evans and Parker have been continuing their 
studies of the malignant transformation of cells in 
culture. Cultures of kidney tissue from 3-day-old 
mouse becomes malignant when cultured no more 
than 145 days in vitro. This indicates that early 
malignant transformation is not restricted to em- 



bryonic tissue in culture. It must now be deter- 
mined whether cells explanted directly into chemi- 
cally defined media become malignant and how 
soon. Tissues grown in diffusion chambers for 
18 months did not produce tumors but did, after 
23 months in the chambers. In collaboration with 
Dr. Yosida the chromosome picture of these cells 
is being followed through this malignant trans- 
formation. He has found that cells remain pre- 
dominantly diploid up to 7 clays in culture after 
which there is an increase in polyploid cells. 

Bryant and coworkers have been studying a 
fragile strain of monkey kidney cells in culture. 
Mechanical or flow stresses were much less on 
these cells in suspension cultures in smaller flasks. 
The cells grew in 125 ml flasks but in 500 ml flasks 
they grew at a much slower rate or not at all. 
They grew better in stationary cultures than in 
the suspension cultures. They also have studied 
binding of methylcellulose molecules to these cells. 
This protects the cells not only in suspension cul- 
ture, but also in the large stationary cultures, pos- 
sibly when they are scraped from the floor of the 
flask. Glucose utilization and lactic acid produc- 
tion of these monkey kidney cells in suspension 
cultures was relatively high. Even in nonprolif er- 
ating cultures the cells were metabolizing actively. 
In proliferating cultures the rate of glucose utili- 
zation was not directly correlated with growth 
rate, but in none of the cultures was the amount 
of glucose remaining in the used fluid so low as 
to be a limiting factor. These cells were also used 
in work with Dr. Montes de Oca on the damaging 
effect of X-irradiation. It was found that serum 
added to the culture immediately before or direct- 
ly after irradiation lessened the damaging effect. 

In the study of preservation of large stocks of 
cultured cells in both serum-free and serum-con- 
taining media, both types have now been success- 
fully preserved by freezing. In collaborative work 
with Dr. Deringer, Dr. Evans is attempting to 
freeze fertilize mouse ova for future implanta- 
tion. 

Genetics 

Studies of effects of specific genes on occurrence 
of tumors are continuing. Heston and Vlahakis 
have now shown that the lethal yellow (A y ) gene 
of the mouse increased the incidence and average 
number of both epidermoid carcinomas and papil- 
lomas induced in the skin by painting with meth- 



NATIONAL CANCER INSTITUTE 



33 



ylcholanthrene. It also decreased the latent period 
of the papillomas. Deringer has shown that the 
A y gene increases the occurrence or reduces the 
latent period of reticular neoplasms. Formerly 
this gene had been shown to increase the occurrence 
of pulmonary tumors and hepatomas and greatly 
decrease the average tumor age of mammary tu- 
mors. Results of transplantation of ovaries in- 
dicate that the effect of the gene on mammary tu- 
mors is not manifest through the ovary. Studies 
are underway to see if this action of the gene is 
through the hypophysis or localized in the mam- 
mary gland itself. 

The effect of the genetic difference between 
strains A and C3H both of which have a relative- 
ly high incidence of mammary tumors in breeding 
females with the virus is revealed when the virus 
is absent. Whereas the tumor incidence in virus- 
free breeding C3Hf females is about 30 percent, in 
a new line of virus-free Af mice not one mammary 
tumor has yet appeared. 

Biochemical Genetics 

Interest has increased in biochemical genetics 
of the mouse. In the Laboratory of Biology it 
is represented by such work as that of Dr. Potter 
and collaborators on proteins associated with plas- 
ma cell tumors and mouse urinary proteins; the 
work of Heston and Rechcigl on catalase ; and the 
nutritional differences in cells in culture dis- 
cussed in the preceding section. 

Dr. Potter has transplanted over 100 plasma cell 
neoplasms in the mouse and has characterized the 
associated protein abnormalities. Seventeen dif- 
ferent Bence-Jones proteins associated with these 
tumors have been characterized. Each Bence 
Jones protein has a characteristic peptide that is 
a stable heritable characteristic of the neoplasm. 
Effort is directed toward determination of number 
of different chains (i.e., gene product units) and 
whether the different peptide maps are a function 
of different disulfide linkages within one molecule. 
Resolution of the question should provide chemical 
information on antibody formation since the pep- 
tides of Bence Jones proteins are similar to those 
in antibody molecules. 

In collaboration with Dr. Finlayson, Potter has 
been studying mouse urinary proteins (MUP). 
They have shown that inbred strains differ in their 
urinary proteins and these are genetically con- 
trolled. 

705-685—63 4 



In the study that Heston, in collaboration with 
Dr. Rechcigl, is carrying out on catalase activity 
of the liver and kidney of mice, a survey of vari- 
ous strains has been made. All strains are approx- 
imately the same in respect to level of kidney cat- 
alase and all strains show a sex difference with the 
level in males being higher than in females. But a 
distinct strain difference has been noted in level of 
liver catalase, strains derived from the original 
Lathrop stock (C57BL, C57BR, C57L, and C58) 
having about half as much as other strains. How- 
ever, the C57BL/An substrain and the C57BL/He 
derived from C57BL/An have a high level. 
Genetic analysis of the difference between 
C57BL/He (high) and C57BL/6 (low) indicates 
that the difference is due to a single gene with low 
level dominant over high. This means that what 
is actually controlled by the gene must be some- 
thing like an inhibitor holding down the level of 
catalase activity in the liver. The original 
Lathrop stock must have had the dominant muta- 
tion for low and a reverse mutation to the recessive 
gene for high must have occurred in the progen- 
itors of the C57BL/An substrain. 

Pathogenesis 

Dr. Mclntire has continued his studies on the 
pathogenesis and pathophysiology of plasma cell 
tumors in mice and man, and has made a detailed 
study of the myeloma kidney in mice associated 
with these tumors. The characteristic lesion is the 
clear, homogeneous, eosinophilic hyaline cast with- 
in the lumina of the renal tubules. There is a con- 
sistency of pattern for a single tumor that would 
indicate that factors such as infection, obstruc- 
tion, and blood supply are not so important as the 
specific characteristics of the Bence Jones protein 
in the production of the myeloma kidney. The 
only component in significant concentrations in 
the casts are the Bence Jones proteins, which by 
themselves are sufficient to cause the casts. Cor- 
relation studies of the characteristics of the 
plasma cell neoplasms and their associated renal 
damage indicate that those tumors associated with 
only a small amount of renal damage have a 
greater amount of cytoplasm. Bence Jones pro- 
teins that produce the least renal damage are those 
that do not precipitate when heated to 56° C. 
Some of the Bence Jones proteins are in the form 
of glycoproteins and this, too, seems to decrease 
their ability to cause renal damage. 



34 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Drug Resistance 



In continuing studies on azaserine-sensitive and 
-resistant lines of plasma cell neoplasm 70429, Dr. 
Anderson has studied the effects of azaserine, 
DON, and the structurally similar duazomycin at 
four steps (glutamine-requiring reactions) in the 
purine synthetic pathway. Three resistant tumor 
lines showed marked cross resistance to duazomy- 
cin as well as to DON. These results character- 
ized duazomycin as probably a glutamine antago- 
nist. It exerted inhibition on all these glutamine 
catalyzed reactions much like DON and possibly 
less like azaserine. In exploring effect of azas- 
erine and DON on sensitive and resistant lines at 
lower levels of the inhibitors a difference between 
the sensitive and resistant lines was observed, the 
reactions being inhibited by lower concentrations 
of the antagonists in the sensitive cells than in the 
resistant cells. 

In studying biochemical differences in the me- 
tabolism of sensitive and of fluorouracil- and 
fluorouridine-resistant lines of the mast cell neo- 
plasm P815, resistance has been found to be asso- 
ciated with a marked decrease in the activity of 
uridine kinase which is probably the major mech- 
anism involved in the resistance. 

LABORATORY OF CHEMICAL 
PHARMACOLOGY 

From the standpoint of public health the major 
objectives in cancer continue to be: (1) better 
methods of treatment; (2) widely applicable 
methods of earlier diagnosis; and (3) preventive 
measures. 

Progress toward the achievement of these goals 
requires more knowledge and understanding of 
the many phenomena involved. The facets of 
the malignant diseases are so numerous as to justi- 
fy the many different approaches that have been 
employed by others. The various programs that 
have been carried out over many years by the 
Laboratory Chief and his associates have had as a 
basis the underlying concept that study of chemi- 
cal agents and of chemical processes would be 
useful for chemotherapy, for prevention of car- 
cinogenesis, and for development of laboratory 
methods of early diagnosis. In continuation 
during the past year of long-range investigations, 
there was planned convergence of these basic 



science studies to illuminate selected aspects of 
the immunology, molecular biology, virology, 
and chemotherapy of cancer. 

The methods and materials employed ran a wide 
gamut, cutting across the conventional scientific 
disciplines. They included the methods of im- 
munology, bacteriology, and virology (both bac- 
teriophages, and viruses that induce tumors in 
mammalian cells) ; mammalian cytology in tissue 
cultures and in tumor ascites ; biochemistry (endo- 
toxic polysaccharides, intermediary metabolism as 
affected by anti-cancer agents, and enzymology) ; 
physical chemistry (density gradient centrifuga- 
tion, streaming birefringence, amperometric titra- 
tion, nuclear magnetic resonance and electron para- 
magnetic resonance spectroscopy) ; radioactive 
tracer techniques; methods of inhibition analysis 
of anti-cancer agents in tumor-bearing animals; 
etc. 

During the past year, research on cancer-causing 
virsuses and on cancer immunology was extended. 
All the organizational units of the Laboratory 
contributed to one aspect or another of these sub- 
jects. Highlights of the work are integrated in 
the following pages without separation on the 
basis of administrative Sections. 

Endotoxins 

Progress was made in several directions: over- 
coming the tolerance, induced by a single dose of 
endotoxic polysaccharide, which prevented tumors 
from responding to a second dose; success in the 
passive transfer of immunity to endotoxin ; chemi- 
cal and physicochemical characterization of endo- 
toxic polysaccharide preparations ; preparation of 
biologically potent endotoxin, containing a mini- 
mal amount of bound lipid, in a homogeneous 
state; demonstration of the presence in the mam- 
malian host of enzymes capable of inactivating 
endotoxin ; demonstration of a basic protein from 
liver which inactivated, reversibly, endotoxic poly- 
saccharide; and synthesis of additional polysac- 
charides with functional groups. 

In the investigation (O'Malley, Shear) of the 
nature of the tolerance induced by S. marcesc&as 
polysaccharide ; and of measures designed to over- 
come it so that tumors would repeatedly respond to 
successive doses, the first two reports appeared in 
the December 1962 number of the JNCI. The 
third paper in this series, describing the surmount- 
ing of tolerance by appropriate spacing and size 



NATIONAL CANCER INSTITUTE 



35 



of the second and later doses, has been completed 
for publication. It reports the repeated repres- 
sion of the growth of mouse Sarcoma 37 by each of 
four successive doses. The high mortality accom- 
panying this procedure, especially after the first 
dose, continues under investigation. Deaths from 
a first dose several times the LD 100 have been com- 
pletely prevented by administration of saline. 
These results are now being prepared for publica- 
tion as well as the progress made in the fractiona- 
tion of blood serum, from mice treated with S 
marcescens polysaccharide, to isolate the potent 
tumor-damaging component. 

The immunity engendered by endotoxins is gen- 
erally held to be ineffectual. Most of the pub- 
lished work on endotoxic immunity has been car- 
ried out in the mouse ; the criterion employed has 
been the number of MLD's neutralized by anti- 
sera. However, normal mice are highly refrac- 
tory to the lethal action of endotoxins. The 
prevailing pessimistic view was re-evaluated 
(Landy) in mice rendered sensitive to the lethal 
action of endotoxin by two different methods : 
implantation of Sarcoma 37 (6 days prior to use) ; 
or, vaccination with BCG (ten days before test). 
These treatments greatly enhanced the sensitivity 
of mice to the lethal action of endotoxin so that 
the highly protective effect of rabbit antiserum to 
Salmonella enteriditis was now demonstrable. 
Passive transfer of 1 ml. of antiserum (0.3 nig. 
antibody nitrogen), a half hour prior to challenge 
with massive doses of S. enteriditis endotoxin, 
neutralized as much as 100 LD 50 and 1.000 tumor- 
damaging doses in mice bearing Sarcoma 37; in 
BCG-vaccinated mice this quantity of antiserum 
protected against more than 1,000 LD 50 . Failure 
of previous efforts to appreciate this powerful 
protective effect, of passive transfer of antiserum 
to endotoxin, was thus found to be a consequence 
of the unsuitable experimental conditions which 
had masked the effectiveness. 

Physico-chemical characterization (Oroszlan, 
Mora) of the tumor-necrotizing polysaccharide 
from Serratia marcescens was carried out by ultra- 
centrifugation and zone electrophoresis. Two 
components were identified. The one with the 
higher molecular weight (S' = 8.24) and which 
migrated more slowly in the electrophoretic field 
was identified as the component possessing tumor- 
damaging activity. Further purification of this 
latter component was accomplished by centrif liga- 



tion in a CsCl density gradient. It was found to 
be dissociable with sodium dodecyl sulfate into 
smaller units with a sedimentation constant of 
S' = 0.9. Depolymerization into these subunits, of 
about the size of haptene, was accompanied by loss 
of tumor-damaging activity. Upon removal of 
the dissociating agent, reaggregation took place 
with reappearance of the biological activity. 
These findings indicated that a macromolecular 
complex of critical size, held together by other 
than covalent bonds, is required for the polysac- 
charide to elicit characteristic endotoxic activity. 

Collaborative work (Landy, Ribi) with endo- 
toxic materials from other gram-negative orga- 
nisms has further clarified the relationship of the 
chemical composition of endotoxic polysaccha- 
rides to their biologic activities. The method 
(Ribi) of isolation from Salmonella enteriditis 
of an endotoxin with minimal amounts of lipid 
and of nitrogen was extended to other bacterial 
species to assess the generality of the important 
findings. Viable bacilli {E. colL Bordet. pertus- 
sis, Serr. marcescens) were extracted with water 
saturated with diethyl ether. The crude endo- 
toxin was deproteinized with phenol-water. Then 
the bulk of bound fatty acids and esters was com- 
plexed with LiAHLj and removed. Polysaccha- 
rides were obtained containing only 0.3-0.5 per- 
cent nitrogen and 2-3 percent fatty acid esters. 
These products displayed undiminished potency 
in evoking the host reactions characteristic of bac- 
terial endotoxin. 

The widely -heralded importance of "lipid A" as 
the component in endotoxins which is responsible 
for their characteristic activities thus has not ob- 
tained any support in these careful studies. To 
the contrary, evidence continues to accumulate 
that it is the polysaccharide moiety which is re- 
sponsible for the potency of these complexes. In 
Eibi's preparations a second component was found 
which was immunochemically similar to the hap- 
tenic polysaccharide obtained on acid hydrolysis 
of endotoxin. The content of this material in the 
endotoxin was reduced, by centrifugation, to less 
than one percent as estimated from gel-diffusion 
data. 

Biologically potent endotoxin has thus been ob- 
tained in an essentially homogeneous state, as in- 
dicated from chemical, ultracentrifugal, immuno- 
diffusion, and quantitative precipitation analyses. 
As was indicated by the work in another part of 



36 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the Laboratory (Oroszlan, Mora) discussed ear- 
lier, it appears increasingly likely that the biologi- 
cal effects require a macromolecular structure com- 
posed of polysaccharide units similar to, if not 
identical with, haptene. 

The discovery of host factors which inactivate 
endotoxin was carried further (Landy, Warav- 
dekar, Shear). Enzyme preparations from two 
sources — blood plasma and cell-free homogenates 
from liver — were incubated in vitro with highly 
purified endotoxin; the products were examined 
with immuno- diffusion techniques. With plasma, 
the reaction product acted like the haptene poly- 
saccharide obtained on acid hydrolysis. With the 
enzyme from liver, no lines of precipitation were 
discernible in the agar gel diffusion test; this in- 
dicated that the endotoxin had been degraded in 
a fashion which did not yield haptene. 

By another procedure a fraction was obtained, 
also from rabbit liver, which inactivated endo- 
toxin by a quite different mechanism, viz., by a 
complexing which was reversible (Oroszlan, 
Mora). Material was leached out of intact cells 
of rabbit liver which rendered S. marcescens poly- 
saccharide incapable of damaging Sarcoma 37. 
This factor from liver was in the basic protein 
fraction. Macromolecular interaction between 
these cationic proteins and the anionic endotoxic 
polysaccharide was demonstrated by zone electro- 
phoresis and by metachromasia. Polyglucose sul- 
fate, a synthetic polysaccharide with more strong- 
ly anionic properties than the bacterial one, 
blocked the inactivation. After inactivation of 
endotoxin by the basic protein fraction, the orig- 
inal tumor-damaging potency was restored by 
treatment with polyglucose sulfate. Other experi- 
ments, with cationic derivatives of synthetic poly- 
glucose, confirmed the conclusion that the inactiva- 
tion was a consequence of complex formation, and 
that the complex was dissociable. 

Synthetic polysaccharides (Mora), and deriva- 
tives containing groups which confer basic or 
acidic properties, have already been useful in a 
variety of investigations. Additional derivatives 
have been prepared (Wood) containing various 
functional groups. 

Cancer Immunology 

In addition to basic science investigations in 
immunology, attention was directed during the 



year to several selected aspects of immunology 
which bear directly on the cancer problem. 

The question of antigenic differences between 
malignant and normal tissues was investigated 
further (Leise, Landy) . The report of McKenna 
and Blakemore on the isolation of a distinctive 
antigen from extracts of HeLa cells could not be 
confirmed; the results of complement-fixation, 
hemagglutination, and gel diffusion tests indicated 
that the antigen they reported was not present in 
the HeLa strain used here. 

Our scientists found that the intact HeLa cell 
evokes antibodies even after a short course of im- 
munization (3 injections) . Bacterial endotoxin as 
adjuvant speeded up antibody production. These 
antibodies were demonstrable only with comple- 
ment-fixation. On the other hand, extracts of 
HeLa cells gave rise to antibodies which could be 
demonstrated by hemagglutination and gel diffu- 
sion as well as by complement-fixation. Here, too, 
endotoxin was an effective adjuvant in obtaining a 
quick response. Fewer antibodies were obtained in 
the rabbit with intact HeLa cells than with cell 
extracts. 

In previous work, it had been reported 
(Messineo) that rabbit antisera to deoxyribonu- 
cleoprotein (DNP) from normal human leucocytes 
did not cross-react with DNP from leukemic 
cells. This line of work was continued. DNP pro- 
duced, with rabbit antisera directed against it, only 
one line of precipitation in agar gel diffusion tests, 
suggesting that the DNP preparations were homo- 
geneous. More highly concentrated solutions of 
DNP were prepared, as high as 20 mg. per ml. 
(the approximate limit of solubility). Antisera 
were also prepared with considerably larger 
amounts of DNP than had been used previously. 
The antisera of higher titer, examined in gel dif- 
fusion against the more concentrated solutions of 
DNP, still showed but one line of precipitation, 
indicating the presence of but a single antigenic 
component. 

Rabbit antisera were prepared against homoge- 
nates of leukemic leucocytes (human) and were 
examined in precipitation tests against antigenic 
material from normal and leukemic leucocytes. 
The results indicated that the aforementioned 
immunological reactions of DNP were not attrib- 
utable to extra-nuclear material. 

The so-called natural antibodies have been recog- 



NATIONAL CANCER INSTITUTE 



37 



nized for many years but their origin and proper- 
ties are still but little understood. The assay for 
natural antibody against gram-negative bacteria 
was greatly simplified (Landy, Weidanz). With 
this improved method, further information was 
obtained (Landy, Weidanz, Henthorne, Silver- 
stein) on the presence of natural antibodies in 
several distinctive situations. These included the 
foetus and neonate, especially in those mammalian 
species where the likelihood of maternal transfer 
of antibody is minimal, and in animals maintained 
in a germ-free environment. Among the sera ex- 
amined were those from foetal lambs, newborn 
swine, calves, and sheep. Among the findings was 
presumptive evidence that in the sheep, at least, 
the foetus is capable of synthesizing natural anti- 
body de novo. 

Cytolytic and cytotoxic antibodies are attract- 
ing the attention of an increasing number of in- 
vestigators. A new electronic method of estimat- 
ing such antibodies was devised (Hirata) which 
is both simple and sensitive. Cells damaged by 
the immunologic system of antibody and comple- 
ment are digestible by trypsin; undamaged cells 
are not digested. An electronic particle counter 
then enumerates viable cells but not those digested 
by trypsin. 

This method was employed in a comparison of 
the potencies of antisera to Sarcoma 37. Ascites 
tumor cells were exposed to various dilutions of 
the antisera, plus complement, after which they 
were treated with trypsin. The surviving cells 
were then enumerated with the electronic particle 
counter. The dilutions which lysed 50 percent of 
the cells represented the relative potencies of the 
antisera. This method is capable of application to 
many cellular problems. The particle counter was 
also found (Hirata) useful in objective estimation 
of hemagglutination. 

Where the electronic method is not available, an 
alternative method of cytolytic antibodies can be 
employed. This was developed (Hirata) on the 
basis that: (1) specific antibody plus complement 
render cells non-viable, and hence susceptible to 
depolymerization of their DNA by DNAase; (2) 
native DNA takes up methyl green whereas de- 
polymerized DNA does not. With Sarcoma 37 
ascites cells as a model system, this method was 
found to be as precise as the electronic one. 

The highly important subject of the mechanism 
of antibody synthesis is being advanced by the use 



of experimental cell-free systems in vitro. Such 
a program was initiated (Sussdorf ) at the molecu- 
lar (sub-cellular) level during the past year. The 
system currently employed has yielded results 
which indicate that it is biosynthetically active. 
This line of investigation has reached the stage 
where rapid data accumulation should permit 
interpretations. 

Tumor-Producing Viruses 

In preparation for investigation, on the molecu- 
lar level, of the mechanisms by which viruses 
transform normal mammalian cells into malig- 
nant ones, experience in techniques of virology 
was acquired (Mora and coworkers) in previous 
years with bacteriophages and bacteria. Inacti- 
vation of T 2 coli phage with polyanions and poly- 
cations was reported previously. 

The orientation of the DNA in the intact phage 
head has been examined (Rizvi, Gellert of 
NIAMD) ) by streaming birefringence. Treat- 
ment with polyglucose sulfuric acid disrupted the 
organized DNA molecule. The mechanism was 
similar to that of cadmium cyanide, i.e., the phage 
lost its ability to kill and lyse bacteria but retained 
full ability to attach. It was concluded that the 
proximal tail structure was damaged. 

In work with oncogenic viruses, it is of course 
helpful to purify further the virus preparations. 
Starch agar gel electrophoresis has been developed 
(Oroszlan, O'Connor) for purification of the vari- 
ous strains of polyoma virus. The large (hemag- 
glutinating) and the small (nonhemagglutinat- 
ing) plaque-forming strains were investigated. In 
the case of the large plaque-forming strain, com- 
plete recovery of hemagglutinin and infeetivity 
was obtained hi a sharply separated zone. The 
hemagglutinin of the small plaque strain is known 
to be masked by an inhibitor substance. Separa- 
tion of the hemagglutinin from the inhibitor was 
achieved with starch agar gel electrophoresis after 
heat treatment of the virus. 

A new medium for the separation of viruses by 
density centrif ligation has been found (Oroszlan, 
O'Connor). The polyglucoses prepared here 
(Wood) gave very stable density gradients which 
provided resolution of polyoma virus into two 
components: intact virus, and empty capsules. 
The resolution was superior to that obtained with 
the methods in the literature. Polyglucose, more- 
over, is inert to virus and non-toxic to mammalian 



38 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



cells ; the dialysis prior to assay in tissue culture, 
which is necessary when CsCl gradients are em- 
ployed, is rendered unnecessary. 

In view of the importance of quantitation in 
assays of oncogenic viruses, attention has been 
devoted to elaborating a standardized technique. 
Several beneficial modifications have been intro- 
duced (O'Connor) in the plaque assay of polyoma 
virus in mouse-embryo monolayers. Titers were 
obtained in excellent agreement with those in Dul- 
becco's laboratory on the same stocks. Work has 
also been started (O'Connor) on the propagation 
of the Eauscher virus in tissue culture with the 
aim of developing a quantitative in vitro assay. 

In the course of systematic investigations on 
chemotherapy of animal tumors, unexpected find- 
ings (Goldin and co-workers) indicated that im- 
munologic phenomena were involved in some of 
those situations. The mice used were pure strains 
(DBA, BALB/c) and F x crosses of them; the 
tumors employed were L1210 and leukemias in- 
duced in these mice with either Moloney virus or 
Eauscher virus. Experiments were carried out 
with skin grafts and with normal tissue implants, 
with and without X-irradiation of the tissues and 
of the hosts. Evidence was obtained (Glynn, 
Bianco, Humphre}^) that antigenic differences 
exist in certain of these situations, and that the 
antibodies which form against the implanted 
tumors can enhance the therapeutic effect of the 
antitumor drug administered. x\nalogous findings 
were made with carcinoma 755 in C57B1 mice 
treated with 6-MP; this was shown by blocking 
of the immune response by pretreatment with 
Melphelan. Abrogation of the homograft re- 
sponse was previously obtained with folic acid 
antagonists; this permitted L1210 to grow in 
BALB/c mice. This line of work was extended to 
other tumors (resistant sublines of L1210, Ca 
755) , to other strains of mice, and with other anti- 
tumor agents (6-MP, 6-TG, BW 322 and 323, 
Cytoxan, Melphelan ) . Suppression of the immune 
response has continued to be obtained, in varying 
degree, in these various situations. 

Chemotherapy 

The various lines of investigation in chemo- 
therapy of animal tumors continued to be prose- 
cuted vigorously and extended (Goldin and co- 
workers) in a number of directions. The influence 



of antibodies, contributing to the effect of chemo- 
therapeutic agents, has already been mentioned. 

The scope of this large and varied program is 
indicated by the following: 

Tumors Studied: Leukemia 1210 and many sub- 
lines, resistant to one or another anti-tumor agent, 
developed in this Laboratory. Adenocarcinoma 
755. Spontaneous mammary tumors. Leukemia 
P1534. Plasma cell and Mast Cell tumors. 
Tumors induced by Moloney Virus and by 
Eauscher Virus. 

Mice Employed: Various pure strains, and then- 
Ex hybrids. 

Chemical Agents Examined: Many folic acid 
and purine antagonists, alkylating agents, plant 
products, and newly introduced synthetic com- 
pounds. 

Topics Investigated: Eole of antitumor anti- 
bodies in chemically induced regressions; chemi- 
cal repression of the immune response; central 
nervous system leukemia; relative efficacy of dif- 
ferent dosage schedules of drug administration; 
combination therapy; drug resistant variants of 
sensitive transpalted tumors; virus-induced leu- 
kemias, and transplantable lines derived from 
them; antiviral chemotherapy; mechanisms of 
drug action and of resistance to therapy (Dihy- 
drofolic reductase; Carbamylphosphate synthe- 
tase; Lactic dehydrogenases) ; evaluation of effi- 
cacy of anti-tumor drugs; selection of compounds 
for clinical study. 

Team Work and Collaborative Work: The 
members of the staff of the Biochemical Pharma- 
cology Section operate as a harmonious team, 
working together in group attack on the various 
facets of the chemotherapy program. (This pre- 
cluded a simple way of indicating the individuals 
working on each of the many lines of work.) Inti- 
mate liaison is maintained with the clinicians 
studying chemotherapy in the cancer patient in 
our Clinical Center. Active collaboration is con- 
ducted with leading scientists in outside institu- 
tions. The Drug Development and Evaluation 
Program (Contract No. PH-43-62-182) is con- 
ducted with the Project Officer (Goldin) and As- 
sistant Project Officer (Venditti) dovetailing the 
project operation with the program within the 
Biochemical Pharmacology Section. 

These activities have resulted in a steady in- 
crease in our knowledge and understanding of ex- 



NATIONAL CANCER INSTITUTE 



39 



perimental chemotherapy in many directions. 
For example, a new class of drugs — the tereph- 
thanilides — has been found to produce longer sur- 
vival of leukemic mice than amethopterin. Quan- 
titative comparisons have been made, in mice with 
advanced leukemia, of six clinically active drugs ; 
these included Vincristine and Methyl-GAG. 
These data were of value for the new Leukemia 
Task Force which sponsored acquisition of infor- 
mation on the correlation of animal data with 
clinical results. Surgical removal of spontaneous 
mammary tumors in C3H mice was followed by 
recurrence in 80% of the cases; with surgery plus 
Cytoxan therapy, recurrences were reduced to 
10%. An important extension of the work con- 
sisted of inclusion of chemotherapy against virus- 
induced tumors and transplantable whole-cell tu- 
mors derived from them, and chemotherapy 
against the viruses themselves. Four compounds 
were tested against inti-acerebrally-inoculated 
L1210; hydroxyurea and methylglyoxal-bis-guan- 
ylhydrazone increased median survival time over 
the controls by 80 and 70%, respectively. Four- 
teen pairs of compounds were evaluated for ability 
to provide therapeutic synergism in the treatment 
of advanced leukemia ; at their optimal combina- 
tion dosage levels, five of the pairs were more 
effective than the optimal daily dose of either drug 
alone. In the treatment of advanced mouse plas- 
ma cell tumor LPC-1, four drugs were found to 
increase survival time as compared with untreated 
controls. 

A battery of six transplantable virus-induced 
lines of leukemia is being developed for large-scale 
assay of antitumor agents. This is required be- 
cause such tumors exhibit a wide range of sensi- 
tivity to chemotherapeutic agents. The six clini- 
cally active drugs mentioned above were rated, 
also, with regard to their effectiveness in mice 
bearing virus-induced transplanted tumors; Cy- 
toxan was the most effective in those experiments. 
A large number (52) of chemical agents is being 
screened in an antiviral assay system. These ex- 
periments are still in progress but, already, the 
technique is being modified by starting with still 
younger mice in an effort to shorten the latent 
period of the virus induced leukemia. Various 
other assay methods for investigations on virus 
tumors are in course of development. With pri- 
mary leukemia induced by the Eauscher virus, 4 
of 11 drugs examined produced an increase in sur- 



vival of more than 100%. Chemicals were tested 
for antiviral activity both m vitro and in vivo. 
Alkylating agents exhibited antiviral activity in 
the former situation ; of seven compounds tested in 
the latter system, Vincristine was found the most 
effective. 

Biochemical studies of the mechanisms of drug 
action, and of resistance to chemotherapy, were 
continued. Attention was focused on dihydrofolic 
reductase activity as a measure of resistance. Met- 
abolite-antimetabolite relationships were investi- 
gated by searching for metabolites capable of re- 
versing the anti-leukemic potency of selected anti- 
metabolites. Other avenues explored involved : the 
elevation of carbamylphosphate synthetase activity 
in livers of mice receiving antileukemic levels of 
hydroxyurea ; lactic dehydrogenases in normal and 
malignant tissues ; the effects of antitumor agents 
on amino acid transport into Sarcoma 37 ascitic 
cells; the association of chromosome alteration 
with increased levels of dihydrofolic reductase in 
amethopterin-resistant variants of L1210 ; the dis- 
tribution of C 14 -labeled Methyl-GAG in sensitive 
and in Methyl-GAG-resistant L1210 ; biochemical 
indices of chemotherapeutic effectiveness; and, 
correlation of therapy against a plasma cell tumor 
with disappearance of the abnormal protein asso- 
ciated with advanced stages of this disease. 

LABORATORY OF PATHOLOGY 

The work in the Laboratory is not restricted to 
a single project, or to a group of closely related 
projects, but each pathologist follows his particu- 
lar line of interest and training. It is, therefore, 
convenient to divide this summary into a number 
of sections. 

1. Collaborative Research 

It is recognized that many research projects at 
the National Cancer Institute require the collab- 
oration of a pathologist, especially in the final 
evaluation of the effect of an experimental pro- 
cedure on laboratory animals. The Laboratory 
of Pathology has always tried to make this assist- 
ance available. The pathologist may take an ac- 
tive part in planning an experiment and in follow- 
ing it through ; he may take the responsibility for 
all autopsies and histologic diagnoses in an ex- 
periment ; he may review only the histologic sec- 
tions in a given experiment; he may serve as a 



40 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



consultant to review selected material with no 
responsibility for the entire experiment or its pub- 
lication. Finally, he may make use of material 
accumulated by other investigators for independ- 
ent studies concerning pathologic alterations. It 
is emphasized that full collaboration of the pathol- 
ogist at the time the experiment is planned is the 
most satisfactory arrangement for it insures the 
best and most economical selection of material for 
pathologic studies. 

In addition to the use of the light microscope 
and standard autopsy procedure, individual mem- 
bers of the Laboratory of Pathology have become 
proficient in special techniques such as fluorescent 
antibody visualization, electron microscopy, tis- 
sue culture, autoradiography, microincineration, 
exfoliative cytology, special cytology, and histo- 
chemistry. These special skills are often used in 
collaborative studies. 

It would be tedious to consider all the collab- 
orative work now in progress in the Laboratory 
of Pathology, especially since it is often covered 
in reports from other laboratories. However, the 
following are noteworthy: Fluorescent antibody 
studies by Dr. Malmgren: (a) plasma cell neo- 
plasms in man and animals carried out with Dr. 
Fahey and Dr. Soloman; (b) relationship of auto- 
immune diseases to sclerosis of blood vessels with 
Drs. Elmore and SokolofF, and (c) location of 
SV 40 virus in cells in tissue culture with Dr. Bab- 
son, Dr. O 'Conor, and Dr. Kirschstein. 

Dr. Malmgren has used cytodiagnosis techniques 
in collaborative studies of metastasis of animal 
tumors with Dr. Madden, Dr. Eetick, and Dr. 
Sabesin. It was found that after an intravenous 
injection of a large number of tumor cells, most of 
the cells had disappeared from the circulation in 
1 minute and all were gone in 10 ten minutes. 
Homologous and isologous tumor cells were re- 
moved from the circulation at the same rate and 
the metastatic potential of the tumor did not in- 
fluence the rate of removal from the blood stream. 
Cytologic techniques were also used in collabora- 
tion with Drs. Eetick, Sabesin, Ketcham and Mrs. 
Hume to study the transplacental transmission of 
melanoma in animals. It was observed that in a 
few instances melanoma cells were found in the 
blood of new born mice whose mothers had received 
an intravenous injection of S-91 melanoma cells. 
Dr. Boss MacCardle in collaboration with Dr. 
Frederick Bartter has described hyperplasia of 



the juxtaglomerular apparatus that is peculiar to 
a condition of hyperaldosteronism and hypokale- 
mia with normal blood pressure. Dr. MacCardle 
has also collaborated with Dr. Van Scott in show- 
ing aborative mitosis during the resting phase of 
the hair growth cycle. 

Dr. Clyde Dawe has collaborated with Dr. 
Helen Curth and grown a human tumor in tissue 
culture. This tumor was associated with acantho- 
sis nigricans in man, but growth in the hamster 
cheek pouch failed to induce skin changes. 

Dr. H. L. Stewart and Dr. K. C. Snell continue 
collaborative work with Dr. Harold Morris on 
tumors induced in rats by several compounds of 
the fluorenamine class; one of these N,N'-2-7- 
fluoroenylenebisacetamide, is remarkable because 
of the great variety of tumors induced. 

Dr. T. B. Dunn has collaborated with Dr. An- 
dervont on a study of tumors in wild house mice ; 
a great variety were found in mice over two years 
of age. Dr. Dunn also collaborates with members 
of the Laboratory of Viral Oncology on the induc- 
tion of tumors in mice by viruses. 

Dr. Elizabeth Chu is collaborating with Dr. 
Eichard J. Wurtman (Laboratory of Clinical Sci- 
ence, NIMH) in the investigation of the relation- 
ship between the pineal gland and the estrous cycle 
of rats. Injection of an extract of the pineal gland 
alters the cycle, and disturbs the hormonal bal- 
ance. Such conditions are always of interest in 
cancer research. 

2. Accumulation of Data Relating to Laboratory 
Animals 

This is a continuing activity in the Laboratory 
of Pathology. Precise knowledge regarding the 
normal anatomy of the laboratory animals is often 
lacking, particularly as regards variations in in- 
bred strains and the alterations appearing when 
animals reach the age when cancer can be ex- 
pected. The investigator using laboratory animals 
must know his basic material for he relies upon it 
much as a chemist relies upon the substrates in a 
reaction. Dr. Stewart and Dr. Snell are accumu- 
lating data on aged rats from five inbred strains. 
Dr. Snell is also collecting data on the Mastomys, 
a species of rodent recently introduced to the Lab- 
oratory. Dr. O'Gara is acquiring information on 
spontaneous diseases of monkeys, especially the 
newborn. Dr. Banfield, Dr. Dunham, Dr. Her- 
rold. Dr. Chus, and Dr. Swarm are collecting data 



NATIONAL CANCER INSTITUTE 



41 



on the hamster, a species now popular in cancer 
research, and about which our knowledge is still 
limited. Dr. Dunn continues to collect informa- 
tion on the endocrine system of the mouse. Dr. 
Swarm has under observation a breed of rats with 
an inborn error in the metabolism of bilirubin. 
Basic data are required before many of these ani- 
mals can be used with greatest profit. 

3. The Natural History of Cancer in Man and 
Applications to Research With Animals 

Pathologists, even when doing animal research, 
remain aware that cancer is a leading cause of 
death in man. They are acquainted with cancer 
in man and rely upon this knowledge for impor- 
tant information and leads for animal research. 
Several studies are now in progress on cancer in 
various ethnic groups in different geographic 
areas. Animal studies have also been devised, 
and are being carried out in the Laboratory of 
Pathology to correlate with observations in man. 
The incidence of cervical cancer in women is being 
studied by Dr. Dunham and others, and Dr. Eliza- 
beth Chu is inducing carcinoma of the cervix in 
the Syrian hamster and noting factors which will 
alter the effect of carcinogens. Dr. Katherine 
Harrold is reviewing lung cancer in veterans of 
World War I, and has devised a method for in- 
ducing lung cancers in hamsters. Dr. Roger 
O'Gara has studied esophageal cancer in natives 
of the Transkei, and obtained an iron pot used by 
victims of this disease, and mice have been exposed 
to metal from this pot as a test for a possible car- 
cinogen. Dr. Lucia Dunham has duplicated the 
quid used by beta! nut chewers who often develop 
cancer, and inserted it into the cheek pouch of 
hamsters, but no cancer has yet resulted. Dr. 
Katherine Harrold is carrying out studies on the 
hamster cheek pouch with known carcinogens. A 
cooperative study on gastric cancer in Japan has 
been started with Mr. Haenszel of the National 
Cancer Institute and Dr. Mitsuo Segi of Japan. 
Urinary bladder cancer is especially frequent in 
New Orleans, and plans have been made to obtain 
adsorbates of the drinking water to test on labora- 
tory animals. Because bladder cancer in Egypt 
appears directly related to schistosoma hemato- 
bium infection, Dr. Louis Thomas has infected a 
large number of hamsters with this organism. No 
bladder cancers have been found. 

An important study on the natural history of 



cancer is being carried out by Dr. Mearl Stanton 
and collaborators. This is the "cancer eye" ap- 
pearing in some cattle herds. This form of cancer 
is of economic importance and may also furnish 
important information on the natural occurrence 
of cancer. Many lesions do not progress to can- 
cer and during incipient stages those that will 
progress cannot be distinguished from the others. 
The lesions begin on nonpigmented areas of the 
lid and the possibility of an insect-borne virus is 
considered. 

4. Carcinogenesis 

Carcinogenesis continues to be a fruitful field 
for study by the pathologist since pathogenesis is 
always of major interest. Types of carcinogenesis 
now under study may be divided into (a) Viral, 
(b) Chemical, (c) Endocrine, and (d) Others. 

(a) Viral carcinogenesis studies with polyoma 
virus 

Dr. Dawe has worked for several years in col- 
laboration with Dr. Law, Dr. Rowe, and Dr. Rab- 
son. Recent findings are that the transformations 
produced by the virus in organ cultures are similar 
to those produced in vivo and transfer from tissue 
cultures back to the mouse produce typical polyo- 
ma type tumors; tissue cultures made from old 
animals react to the virus as readily as cells from 
young animals; total body irradiation makes 
adults susceptible to induction of tumors ; specific 
antipolyoma antibody if used before the 5th day 
of a culture infected with polyoma will prevent 
tumor formation; and epithelium does not show 
neoplastic poliferation in the absence of me- 
senchyme, in fact a combination of both elements 
is required. It is expected that time lapse pho- 
tography will help to elucidate this perplexing 
problem of epithelial-mesenchymal association. 
Discovery of this association raises important 
problems relating to embryogenesis and tumori- 
genesis. Dr. Mearl Stanton with Dr. H. Otsuke, 
a Visiting Scientist, has continued studies on poly- 
oma-induced hamster tumors. Dr. Alan Rabson 
has produced a variant of polyoma with reduced 
oncogenic potency by growing it on a milk me- 
dium. Dr. Robert Friedman has found a reduced 
interferon production in this strain. He has also 
been studying the role of interferon in recovery 
from viral infections and the possible therapeutic 
use of agents that stimulate interferon production. 



42 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Dr. Malmgren and Dr. Rabson found that infec- 
tion with polyoma virus did not affect chemical 
carcinogenesis. 

Studies have also been conducted with SV 40 
virus, which produces tumors in hamsters. Dr. 
Dawe observed transformation of tumor kidney 
cells in tissue culture by this agent. Ependy- 
momas in Mastomys were produced by Dr. Rabson 
and coworkers by the SV 40 virus, and morpho- 
logic changes were produced in human thyroid 
cells in culture. Dr. O'Conor is now studying the 
polyoma and SV 40 virus with the electron micro- 
scope. Dr. Dunn has continued collaborative 
work on the leukemia-inducing viruses of Dr. 
Moloney and Dr. Rauscher. An experiment with 
the Rauscher virus in BALB/c mice failed to in- 
duce leukemia, probably because the animals died 
early from hemorrhages in the spleen. An experi- 
ment now in progress with a hybrid mouse should 
be more successful since reaction in spleen is less 
severe. Dr. Kamel has examined over 1,000 
BALB/c mice injected with material from human 
cancer. Material from one gastric carcinoma 
caused an increase in renal tumors that appears 
to be significant. 

(b) Chemical carcinogenesis 

Some of these studies have already been referred 
to in this summary, because they were attempts to 
duplicate conditions that might appear in man. 
Dr. Mearl Stanton has produced lung cancer in 
rats by a combination of an infarct-producing 
chemical and methylchloanthrene. Other poten- 
tial carcinogens including viruses are now being 
investigated, especially those implicated in human 
lung cancer. This experimental device offers 
many possibilities and represents a combination of 
factors which may simulate conditions in human 
carcinogenesis. Dr. Roger O'Gara and collabora- 
tors are investigating two interesting and impor- 
tant facets of chemical carcinogenesis. (1) Induc- 
tion of neoplasms by minute doses of a carcinogen 
given to newborn animals ; the great susceptibility 
of the newborn animal indicates that special pre- 
cautions should be taken to avoid exposure in the 
young human being. (2) Attempts at induction 
of cancer in newborn monkeys have not yet suc- 
ceeded. However, much needed experience and 
information is being acquired on care of the young 
primate, and diagnosis of its diseases. Dr. Am- 
badas Mulay has continued his studies on the in- 



duction of hepatomas in rats and substances that 
affect the growth of these tumors. Dr. Dunn has 
found a high percentage of cancers of the vagina 
in mice given a single dose of diethylstilbestrol at 
birth, and kept 12-26 months. Three granular 
cell myoblastomas were also found and one has 
been successfully transplanted. Dr. O'Gara has 
attempted to induce epithelial thymic tumors in 
mice by stilbestrol and radiation, but although 
lymphosarcomas are induced true thymomas have 
not j^et appeared. Early results indicate that mice 
thymectomized when newborn have developed 
more tumors from carcinogen injection that sham 
operated controls but the results are not final. 
This work is being done in collaboration with Dr. 
Malmgren. Dr. Richard Swarm is studying 
thorium induced neoplasia in man and in experi- 
mental animals. 

5. Transplantable Tumors 

The first studies on transplantable tumors in 
animals were made by pathologists and this in- 
terest has continued. The interpretation of the 
effects of certain tumors on the host has given im- 
portant information regarding the function of 
normal organs, especially the endocrine organs. 
Work on transplantable adrenal cortical carcino- 
mas is being continued by Dr. Mulay. Dr. Mulay 
has found an absence of glucocorticoid secretion 
by one of these tumors. Dr. Swarm has found that 
the uptake of S 35 in a chondrosarcoma is reduced 
when compared with normal cartilage of the host. 
Dr. Swarm is also investigating the transplanta- 
tion behavior of this tumor in comparison with 
normal cartilage. Dr. Banfield, with Mrs. Brind- 
dley, has continued to study a contagious reticu- 
lum cell sarcoma in the hamster. There is some 
indication that contagion is not always by cellular 
transfer because some animal contacts have devel- 
oped tumor 17-19 months later. Dr. MacCardle 
has made further studies on freshly-induced and 
transplantable plasma cell tumors. The same type 
of cell, the plasma cell, produces different types of 
globulin at different stages of maturation. Dr. 
El Bolkainy, a Visiting Scientist, has studied a 
hormonally active adrenal cortical tumor in the 
mouse. He has developed methods for hypophy- 
sectomy and observed variation in the tumor when 
grown in hormonally altered hosts. This work 
will be continued when he goes to the University 
of Michigan for postgraduate work. 



NATIONAL CANCER INSTITUTE 



43 



l:IOOO 



ABDOMEN 



DAYS 



4 + 



nun in 



3 + 



iiiiiii ii 



2 + 



i in mi i 



I +■ 



i mill 1 1 







i iiiiiiii 



4-h 



3 + 



ii 1 1 1 in i 1 1 mi ii i 



i iniiii i 



1:3000 



2 + 



1 1 1 1 1 1 1 1 1 



28 



iiiiiiii 



^ a 




m 



a 



a 



^_^ 



^ 



^ 



^ 



DAYS 



1: 1000 



Chart la 

BACK 



1:3000 




Chart lb 
Age variations in the acetic acid-extraetable collagen from hamster skin 



One-tenth of a gram of skin, kept in a single piece as far as 
possible, was extracted with 5 cc. of dilute acetic acid (1 : 1000 
or 1 : 3000) for 24 hours at about 9° C. About 1.5 cc. of the ex- 
tract was then transferred to a 10X7.") nun. test tube and about 
0.3 gm. of sodium chloride crystals added. If soluble collagen was 
present, it precipitated in characteristic white fibrils, which were 
lighter than the extracting medium and tended to float to the top 
of the tube. The precipitate was graded visually and estimated 
at to 4-f. A few strands of precipitate floating to the top of the 
tube were graded 14- ; a veil over the salt subsequently floating 
to the top. 2 4-, and a heavy precipitate sticking to the salt. 
3 + . A concentration of collagen in solution great enough to form 
gel around the salt and causing all the granules to clump was read 
as 4 + . 



A portion of the initial extract was diluted 1 : 8 and the pre- 
cipiate measured again to obtain a finer measure of the amount 
of collagen extracted. 

Animal age in days is in the left hand column. The results for 
extraction using 1 : 1000 dilution of acetic acid arc on the left of 
the chart and those for 1 : 3000 dilution on the right. The bars 
represent the number of animals of each age whose skin extract 
gave the precipitate designated by the plus reading at the top of 
each column. Each mark below the top line represents one ani- 
mal. The solid bars represent the undiluted extracts and the 
hatched bars the extracts diluted 1 : 8. Chart 1-a is for ab- 
dominal skin and 1-b for back skiu. 



44 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 

■JS«sr 







#~ « ***** v*^ 






* 





FIGURE 1. — Normal elastic fibers. Papillary layer abdomen, 6- 

year-old W.M., Acute leukemia. Autopsy. X 7200. 
Figure 2. — Normal elastic fibers. Skin, abdomen, 21-year-old 

W.M., Tetrology of Fallot. Autopsy. X 7200. 
Figure 3. — Normal elastic fibers. Note prominence of fibrils and 

density of matrix. Papillary layer buttock, 61-year-old W.M., 

mycosis fungoides. Clinic. X 7200. 
Figure 4. — Normal elastic fibers. Note peripheral arrangement 

of fibrils. Recticular layer buttock, 75-year-old W.F., mycosis 

fungoides. Clinic. X 7200. 



Figure 5. — Probably normal elastic fibers which appear exploded. 
This may be an artifact. Papillary layer buttock, 56-year-old 
W.M., psoriasis. Clinic. X 7200. 

Figure 6. — Elastotic degeneration. Note the large size of the 
fibrils in the otherwise normal-appearing portions of the elastic 
fiber. There is a transition within the same fiber to more ad- 
vanced degeneration with an increase in density of the matrix 
which begins to appear very finely granular. 



NATIONAL CANCER INSTITUTE 



45 



6. Miscellaneous 

(a) Dr. Louis B. Thomas has studied the intra- 
cerebral localization of leukemic L1210 cells in 
mice treated by several agents. A remarkable cor- 
relation with children treated for acute leukemia 
has been found. As a result of this work, impor- 
tant information on the spread of leukemic cells 
and a better understanding- of the anatomy of 
the brain of the mouse has also been obtained. 

(b) Dr. Richard Swarm continues his work 
with hyperbilirubinemic rats. It is desirable that 
some degree of inbreeding be achieved so that they 
will accept tumor transfers. 

(c) The aging process in collagenous and elas- 
tic connective tissue has been studied by Dr. Ban- 
field. Much fundamental information has been 
acquired on these important body components. 
During an electron microscopic investigation of 
senile elastosis observations on age changes in the 
elastic fibers were made, best brought out in the 
accompanying figures 1-4. 

The observations on senile elastosis can be sum- 
marized as follows : 

The elastotic degenerated material in senile 
elastosis was found to be derived from elastic 
fibers. There was no definite evidence that a 
change in collagen could also result in elastotic 
degenerated material. Observations on normal 
elastic fibers incidental to the problem of senile 
elastosis indicated that the possibility of a normal 
collagen-elastin transformation should not be 
dismissed. 

The acetic acid solubility of the skin collagen in 
aging hamsters has been followed from 28-809 
days and is summarized in the accompanying 
chart. Here only the data for the transition 
points have been charted. Organ weights and 
body weights were also recorded and regression 
lines are being plotted. With one exception, there 
is no obvious correlation of age changes in the 
organs or pathology of the organs with the col- 
lagen solubility. The exception, a slight increase 
in solubility at the maturation of the testis after 
28 days. 

(d) Dr. Louis Thomas is a member of the Com- 
mittee on Nomenclature and Classification of Dis- 
ease of the College of American Pathologists. 
Classification of neoplasms is a particularly diffi- 
cult field. 



(e) Dr. Boss MacCardle is engaged in writing 
a monograph on the cytological characteristics of 
a variety of neoplasms. He is also writing the 
Introductory Chapter for the new revision of 
Cytology and Cell Physiology, by G. H. Bourne, 
to be published by the Academic Press. 

LABORATORY OF PHYSIOLOGY 

Excellent progress has been made by Dr. Pratt in 
developing mathematical models and statistical 
techniques for evaluating cancer chemotherapy 
screening data, particularly in the analysis of 
structure-activity relationships of alkylating 
agents in a spectrum of rat tumors. A method 
was formulated for creating a file of pathological 
diagnoses and vital statistics in a form available 
for selective retrieval. By employing an IBM 
computer 1620, programs have been prepared and 
tested in which it was possible to demonstrate an 
ability to retrieve data either on a single or multi- 
ple selection basis. The program started last year 
by Dr. Pratt in collaboration with Drs. Rushizky 
and Sober of the Laboratory of Biochemistry to 
develop chemical, mathematical and computer pro- 
gramming techniques to describe the arrangement 
of mononucleotides within polynucleotides from 
yeast RNA has made considerable progress. Mono- 
nucleotides of adenine, guanine, cytosine and 
uracil from yeast RNA, prepared by alkaline 
hydrolysis or enzymatic methods, show mathemati- 
cally similar identity to their counterparts from 
other sources, including tobacco mosaic virus RNA, 
micrococcal RNA or polyadenylic acid. Using the 
four above-mentioned nucleotides (Ap, Cp, Gp 
and Up) in their library, they have been able to 
mathematically describe, identify and obtain base 
ratios for di-, tri-, and tetranucleotides of known 
chemical identity (e.g., ApCp, CpApGp, 
GpGpUp, UpGp, ApApApAp, etc) . Of particu- 
lar interest are the hyperchromic and hypochromic 
(shifting of spectral maxima) effects observed 
after hydrolysis which appear to be highly charac- 
teristic expressions of near neighbors within the 
polynucleotide. As a result of this technique it 
may be possible to tackle problems of chemical 
structure and sequence that have been too large or 
too laborious to undertake previously. Dr. Pratt 
in conjunction with Drs. F. K. Millar, Seoras D. 
Morrison and Peter Baillie have been investigating 



46 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the total metabolism of the tumor-bearing animal 
with a view of determining the mechanism by 
which the presence of a tumor affects the normal 
patterns of energy and material exchange. They 
hope to study the problem of caloric imbalance in 
the tumor-bearing animal. This involves an 
understanding of the relationships between calo- 
ries, nitrogen, electrolytes and water as these rela- 
tionships change with progressive tumor growth. 
Some of these relationships are being studied in 
animals made hyperphagic or aphagic by hypo- 
thalamic lesions ; a study of temporal patterns of 
food intake in normal intact rats, in rats after gas- 
tric vagotomy, in rats after lesions in the hypo- 
thalamus; temporal patterns of gastric motility 
and its relationship to food intake. The problem 
of ulcer occurrence in tumor-bearing rats is being 
studied with respect to time of appearance and 
tumor development and methods which can be used 
to block their occurrence. 

Characterization of nucleic acids and nucleo- 
proteins of normal and malignant tissues in terms 
of physical, chemical and biological properties as 
well as the development of new procedures for 
the isolation of nucleic acids continue to be ex- 
plored. Dr. Shack has obtained experimental con- 
ditions whereby complete separation of native and 
denatured DNA in the density gradient electro- 
phoresis apparatus is possible. Denaturation ex- 
periments in which DNA is partially denatured 
can be resolved into one fraction which is com- 
pletely native and the other completely denatured. 
These data provide the first definitive proof that 
denaturation of DNA is an all or none phenome- 
non. This strongly suggests that the DNA macro- 
molecule exists either in the native or denatured 
configuration. No evidence was found for inter- 
mediate hybrid species which possess a partly na- 
tive and a partly denatured configuration. Native 
DNA derived from partially denatured material 
has a higher content of guanine and cytosine, and 
a lower adenine and thymine than the original 
total DNA, while the denatured fractions show a 
lower guanine and cytosine and a higher adenine 
and guanine than the original DNA. The extent 
of these deviations depends on the extent of par- 
tial denaturation. 

Dr. Wollman is continuing his program dealing 
with properties of transplantable thyroid tumors, 
the iodide-concentrating mechanisms of the thy- 
roid gland, and the intracellular properties of the 



thyroid gland. He has found that in the large 
family of transplanted thyroid tumors derived 
from feeding rats a goitrogen that transplanted 
independent tumors (those which will grow in 
rats not fed a goitrogen) arose from very small 
regions in the parent donor tumor. Some of the 
functional independent tumors can clear radio- 
iodide from the blood at rates which are independ- 
ent of pituitary production of thyroid-stimulat- 
ing hormone. The rate at which these functional 
tumors clear blood radioiodide indicate a high 
rate of blood flow comparable to rates in the liver 
and brain. Kinetic studies on the distribution of 
radioidide among the iodinated amino acids of 
the thyroid gland in the rat and guinea pig indi- 
cate that I 131 -labeled thyroxine in the gland is 
turned over in the gland much more rapidly than 
it is secreted into the blood. Studies are under- 
way on the properties of intracellular colloid drop- 
lets in the thyroid gland to determine whether 
they are newly formed colloid or colloid which has 
been resorbed from the lumen of the follicle. The 
purpose of this study is to obtain a better under- 
standing of the mechanism of hormone secretion 
and, thus, radioisotope release in the thyroid 
gland. 

Dr. Elkind is pursuing his studies on the mech- 
anisms of survival and recovery of X-irradiated 
mammalian cells in tissue culture. Employing 
tracer counting and radioautographic techniques 
he is also examining the effect of X-irradiation on 
DNA, RNA and protein synthesis. He has ob- 
served that after irradiation the synthesis of DNA, 
RNA and protein continues during the division 
delay period ; the rate of DNA synthesis after ir- 
radiation undergoes fluctuations, probably as a 
result of the combined effects of suppression of 
DNA synthesis in cells undergoing division (S) 
at the time of exposure and the progression of the 
Gi cells (those about to undergo mitosis) into cells 
being synthesized. After a single exposure there 
does not seem to be a temporal relationship be- 
tween the fluctuations in the rate of DNA synthe- 
sis and the fluctuations in survival previously 
observed. He is also developing models for the 
radiological response of cells and tissues. His 
objective is to construct models for a better under- 
standing of radiation effects in tissues. In collab- 
oration with Dr. R. Bellman of the Rand Cor- 
poration and Dr. M. Berman, NIAMD, a Monte 
Carlo model has been programmed for the effect 



NATIONAL CANCER INSTITUTE 



47 



of irradiation on the survival and growth of a 
population of cells. 

Dr. Willie Smith has been engaged in studying 
irradiation injury as well as spontaneous and in- 
duced recovery. She has set as her objectives (1) 
to characterize and quantitate various aspects of 
irradiation injury and recovery, (2) to correlate 
natural factors such as age, weaning, intestinal 
flora on injury and recovery, (3) investigate the 
processes involved in the initiation of recovery by 
treatment with substances such as endotoxin and 
various mitotic inhibitors, and (4) to study the 
effect of mitotic inhibitors on the response of tu- 
mor cells to radiation. Experimental data indi- 
cate that there is a definite relationship between 
radiation dose and granulocyte or lymphocyte 
count three or four days after irradiation. Higher 
dose ranges result in lower counts and an increase 
in the time interval between exposure and recov- 
ery. Young mice (4 weeks) required a longer 
time for recovery although the counts are the same. 
Colchicine-treated mice require less time for re- 
covery although the counts are the same as the 
controls. Velban (a mitotic inhibitor) promotes 
early granulocyte and lymphocyte recovery and 
increase in per cent survival in irradiated mice. 

Dr. Draper has been studying the status of 
antibody- forming mechanisms after multiple ex- 
posures to radiation, as well as investigating the 
action of the antigen and non-specific stimuli dur- 
ing the immune response in irradiated animals. 
Rabbits were irradiated and then immunized with 
various doses of washed sheep erythrocytes. Se- 
rum samples obtained during the response were 
assayed for hemolysin content. The radiation- 
induced depression of hemolysin formation can be 
overcome to a large extent by a second antigen 
injection one day after a second dose of irradia- 
tion. The results indicate that the depression of 
antibody formation by a given dose of high energy 
(2.0-2.5 Mev) X-irradiation is less severe than that 
of lower energy irradiation. The modification of 
depressive effects of irradiation is dependent upon 
the dose of each of the two antigen injections and 
upon the time interval between the two radiation 
exposures and antigen injection. Relatively large 
amounts of antibody are produced in rabbits given 
a total of 1,200 r in two exposures one week apart, 
provided antigen is injected one day after each 
exposure. Dr. Draper is also studying the effect 
of daily, low level gamma irradiation on the 



ability of rabbits to produce antisheep hemolysins. 
Thus far he has demonstrated that the hemolysin 
response following accumulated doses from 100 
to 1,900 r is not markedly depressed. The spleen 
appears to be more radiosensitive to radiation un- 
der these conditions than nonsplenic sites of he- 
molysin synthesis. 

The chemical effect of ionizing radiation on 
aqueous solutions of organic compounds, particu- 
larly acetone, is being studied by Dr. P. Reisz em- 
ploying Co 60 gamma rays. In the present study 
of aqueous acetone solutions, it has been possible 
to distinguish between the chemical effect of the 
solvated election and the hydrogen atom and to 
measure the relative rate constant of these two 
species of acetone. The initial yield of hydrogen 
atoms and solvated elections have been shown to 
be a function of pH. Isopropanol has been identi- 
fied as a new product in addition to those reported 
last year. The mechanism of action is currently 
being investigated and one mechanism consistent 
with the current experimental results has been 
proposed. 

Dr. C. Maxwell is furthering his studies on the 
effect of ionizing radiation on amino acids in aque- 
ous solution. He is actively pursuing the mecha- 
nism of the chemical reactions induced by ionizing 
radiation with the ultimate goal of compiling in- 
formation on simple systems of biological interest 
with the intention of applying these observations 
to more applied complex systems which are not 
amenable by direct investigation. 

Studies of urinary excretion patterns of nucleic 
acid congeners in human leukemia are being con- 
tinued by Dr. J. C. Reid. Because of the complex 
nature of the urinary profile, mathematical as well 
as chemical techniques had to be employed. Chem- 
ical procedures comprise an integrated chromato- 
graphic system developed by Dr. Reid in which a 
urinary specimen can be fractionated into a large 
group of mixed fractions. The mathematical 
techniques, which are primarily those of linear 
algebra, represent an approach to the further reso- 
lution of mixed peaks by means of computational 
analysis of their ultraviolet absorption spectra. 
Data processing machinery is used to handle the 
large volume of data generated. Thus far an inte- 
grated system for data reduction, error monitor- 
ing, retrieval, etc., has been created and is working 
satisfactorily. In addition, a system of programs 
has been written which analyzes unknown mix- 



48 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tures containing as many as eleven components in 
which one of the components can be computed with 
errors as low as a few tenths of one percent. This 
type of mathematical and machine approach 
makes it possible to detect the presence of mean- 
ingful information in such datum which may be 
the ultimate interpretation. 

Studies on protein synthesis in normal and 
tumor cells and the effect of antagonists are being- 
continued by Dr. M. Eabinovitz. His objectives 
are to study in both normal and tumor cells the 
pathway taken by amino acids in the formation 
of protein through an analysis of biochemical le- 
sions introduced within the pathway by specific 
inhibitors of protein synthesis and, where appli- 
cable, to extend the information obtained to the 
tumor-bearing animal with the view of selective 
inhibition of protein synthesis in cancer cells. He 
has found that hemoglobin synthesis can be inhib- 
ited by a-amino-/3-chlorobutyric acid at the earliest 
stages of incubation. Synthesis appears to con- 
tinue for a period of 5 minutes at half the normal 
rate and then stops abruptly. The accumulation 
of labeled protein on the ribosomes, which is char- 
acteristic of this inhibition, is the result of contin- 
ued labeling of ribosomal protein beyond the time 
when normal ribosomes would have reached a 
steady state of amino acid incorporation and pro- 
tein release. The initial rate of labeling of ribo- 
somal protein is identical in normal cells and in 
those treated with the antagonist. The mainte- 
nance of labeled ribosomal protein accumulated in 
the presence of the antagonist requires the func- 
tioning of energy yielding processes of the cells. 
The use of amino acid antagonists in cell-free sys- 
tems which incorporate amino acids into protein 
offers both a means of studying the partial reac- 
tions of the system and can also serve as a basis of 
comparison of the cellular and acellular processes. 
Preliminary studies have indicated that the incor- 
poration of labeled amino acids into protein of iso- 
lated Ehrlich ascites ribosomes is insensitive to in- 
hibitors which interfere with the process by intact 
cells. The results suggest that the acelluar system 
may represent a small insensitive component of the 
total cellular activity. 

With isolated rabbit reticulocytes, a striking 
similarity between the celluar and acellular proc- 
esses was observed. 

Drs. J. White, F. K. Millar and Mrs. J. N. Toal 
have continued their studies of the growth stimu- 



lating factors in tumor tissue and sodium balance 
in tumor-bearing rats with emphasis on the par- 
ticipation of the adrenal gland. A diet containing 
dried tumor tissue (10%) as a source of protein 
produces a 30% better growth (increased over-all 
weight) in normal animals than does a 10% 
casein diet. Changes in adrenals have been ob- 
served in tumor-bearing rats. The zona glo- 
merulosa was 40-50% wider in the adrenals of 
tumor-bearing rats than in the adrenals of normal 
rats. This observation was in line with previously 
reported increase in aldosterone secretion by 
adrenals of the tumor bearers. Studies have been 
initiated on the effect of sublethal does of a X-ir- 
radiation on the incidence of cirrhosis in rats in- 
gesting a low protein diet. Sprague-Dawley rats 
ingesting a 6% casein diet and exposed to 450 r all 
develop cirrhosis of the liver in 145 days. Control 
non-irradiated rats also develop cirrhosis but not 
as severe although no irradiation had been admin- 
istered. Osborne-Mendel rats similarly treated 
develop cirrhosis only in the irradiated group. 
Supplementing the diet with choline or methionine 
prevents the appearance of cirrhosis of all animals 
previously affected. 

LABORATORY OF VIRAL ONCOLOGY 

The research of the Laboratory of Viral On- 
cology continues to be primarily in the area of 
viruses in relation to cancer, although other basic 
problems such as the ultrastructure of normal and 
nonviral cancer cells, cancer immunology, and the 
induction of plasma cell tumors with chemically 
inert substances are also being pursued. 

Dr. Moloney has continued investigations on the 
strain of mouse leukemia virus which he first iso- 
lated from the transplantable tumor of mice, S 37. 
Dr. Moloney has confirmed the work of Sachs in 
which it was shown that the transplantability 
of tumors induced with the Moloney strain of 
mouse leukemia virus is inhibited in isologous 
mice by preinoculation of the leukemia virus into 
recipient hosts. This phenomenon was first ob- 
served for the polyoma mouse-virus system, in- 
dependently, by Habel and co-workers and by 
Klein. The phenomenon is thought to indicate 
that a new cellular antigen is induced by the 
virus in the membranes of the virus-induced tumor 
cells, and that the host is able to react against the 
transplanted cells containing this foreign antigen 



NATIONAL CANCER INSTITUTE 



49 



through immunological factors brought into play 
by the prior inoculation of virus. However, much 
work remains to be done to establish this concept 
as the actual mechanism involved. A possible 
application of this new information is the devel- 
opment of a relatively short-term biological assay 
of the virus. Using the rejection of transplanted 
tumor cells as the biological indicator, and prior 
injection of the candidate virus suspension, quan- 
titative differences in potency up to 1,000-fold 
have been determined by Dr. Moloney in assays 
completed within less than 30 days. This is in 
comparison with a period of 4 months or longer 
required for comparable bio-assays involving leu- 
kemia development as the biological indicator. 
Another potential practical application of the 
phenomenon resides in the possibility that it is 
highly specific for virus strains and may there- 
fore be used for detecting antigenic differences 
between leukemia virus strains. Little progress 
has been made thus far toward this important ob- 
jective because of the failure of conventional 
serological methods to yield satisfactory results 
with the murine leukemia viruses. 

In other studies initiated during the past year 
Dr. Moloney has achieved preliminary success in 
isolating infectious nuclei acid from extracts of 
murine leukemic tissues induced by his strain of 
virus. The method employed was a modification 
of the phenol method of Gierer and Schram which 
others had successfully applied to several viruses. 
The incidence of leukemia in mice which received 
the nucleic acid fraction was 11.8 percent within 
6 months, which is well above the natural incidence 
(less than 1 percent) at this age in the strain of 
mouse employed (BALB/c). The biological ac- 
tivity of the nucleic acid fraction was destroyed 
by RNase but not by DNase. The activity of the 
intact virus, on the other hand, was not altered by 
RNase (or by DNase) . The leukemia induced by 
nucleic acid has been confirmed by Dr. Dunn 
as being identical to that induced by the intact 
virus, and complete viral particles have been ob- 
served by Dr. Dalton in the leukemic cells and 
megakaryocytes of mice with the nucleic acid in- 
duced disease. These results have now been re- 
peated in three out of five tries, with essentially 
the same incidence of tumors. 

In collaborative studies, Drs. Moloney and 
Dunn have found that inoculation of the Moloney 
strain of leukemia virus 48 hours after thymec- 



tomy induces recticulum cell sarcomas (type B) 
in recipient BALB/c mice, rather than lympho- 
cytic neoplasms as in intact hosts. Also, the re- 
ticulum cell tumors appear after a much longer 
latent interval than the lymphocytic neoplasms. 
Splenectomizecl hosts may develop chloroleuke- 
mia, lymphocytic leukemia, or reticulum cell sar- 
comas (type B) as a result of inoculation with 
Moloney's virus strain. 

Collaborative studies by Drs. Moloney, Dalton, 
and Dunn are being conducted on the kinetics of 
virus development and the early pathogenesis of 
the disease in mice and rats inoculated with 
Moloney's strain of virus. Virus particles can be 
detected by electron microscopy in megakaryocytes 
of the bone marrow as early as 7 days after in- 
oculation of the virus, long before any histologi- 
cal evidence of disease can be detected. Relative- 
ly large quantities of virus can be detected after 
14 days. As previously reported, large quantities 
of virus can also be demonstrated in the blood 
within a few weeks after virus inoculation and 
before the appearance of overt disease. This find- 
ing has made possible the production of virus sus- 
pensions of a much higher degree of purity than 
can be achieved when tissue extracts are used as 
the source of virus. Blood contains relatively 
little extraneous particulate material in the virus 
size range and a high degree of purification can 
be obtained by differential centrifugation alone. 

Dr. Dalton, in collaboration with Dr. Moloney 
and Dr. Haguenau (of the Institute of Cancer 
Research, Villejuif , France) has recently initiated 
studies on the morphology of the Moloney strain 
virus utilizing the method of negative staining by 
salts of phosphotungstic acid. Earlier results 
utilizing thin sectioning techniques had indicated 
that some Moloney virus particles possessed tails, 
giving the particles a "tadpolelike" appearance. 
The negative staining of intact virus particles on 
monolayer film preparations revealed a high per- 
centage of particles with tails. In many instances 
an hexagonal geometric structure of the body of 
the virus particle could be made out. Although 
"tails" and other pleomorphic formations of other 
animal viruses have been described as artifacts 
under certain conditions (e.g., drying in the pres- 
ence of strong salts), observance of the tails of 
Moloney's virus in preparations that were fixed 
before drying, in thin sections, and the absence of 
other pleomorphic forms indicate that the tail 



50 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



in this instance is real and that it may represent a 
stage in maturation. This characteristic mor- 
phology in negatively stained material has made 
possible rapid analysis of extracts from blood, 
tissues and tissue culture for the presence of virus 
and has thus greatly opened up studies on kinetics 
of virus reproduction. 

In further studies on the Moloney strain leu- 
kemia virus in tissue culture, Dr. Manaker has 
demonstrated that it can be propagated in cultures 
prepared from mouse lung and kidney, in addition 
to mouse spleen preparations previously reported. 
Whether the virus was propagated in cells of the 
reticulo-endothelial system present in the differ- 
ent organs tested was not determined. 

Other studies by Dr. Manaker indicate that this 
murine leukemia virus does not pi'opagate in cul- 
tures prepared from human or monkey bone mar- 
row, whereas it can be readily propagated in 
mouse bone marrow cultures. This suggests that 
man and other primates may not be susceptible to 
this murine leukemia virus. Mouse tissue cultures 
held at 40° C. continued to release Moloney virus 
without detectable difference from cultures held 
at the usual temperature of 37° C. Efforts by Dr. 
Manaker to infect tissue cultures with nucleic acid 
fractions of virus supplied by Dr. Moloney were 
unsuccessful. Tests on mouse tissue cultures origi- 
nally infected with Moloney strain virus iy 2 years 
ago, and maintained since that time by Dr. Man- 
aker, showed that virus was still being released. 

Dr. Fink has continued her efforts to adapt con- 
ventional serological methods to the Moloney leu- 
kemia virus. Slight complement fixation at low 
titer was achieved but the sensitivity of the sys- 
tem will have to be increased before complement 
fixation becomes a useful tool. All efforts to dem- 
onstrate specific fluorescence in leukemic cells 
using fiuoroscein conjugated antibodies in both 
mouse and rabbit antisera have failed thus far. 
However, in view of the importance of this tool 
to studies on human leukemia, intensive efforts to 
devise adequate methods of immunofluorescence 
for this mouse model system are being continued. 
Exhaustive tests involving numerous controlled 
variables have failed to demonstrate a hemagglu- 
tinin associated with the Moloney virus which 
might permit the establishment of a hemagglu- 
tination test. 

Dr. Rauscher has continued investigations on 
his strain of mouse leukemia virus, the isolation of 



which was reported last year. This virus induces 
a dual type of disease the early phase of which is 
represented by marked erythrocytopoiesis and 
tremendous enlargement of the spleen ; the second 
phase is represented by the development of lymph- 
ocytic leukemia. The early enlargement of the 
spleen (at about 15 days with the strongest does) 
permitted the development of practical bioassay 
procedures based upon time to palpable spleen, or 
weight of spleen at sacrifice. Using these assay 
procedures the virus was found to remain stable 
and show no loss in potency when stored nine 
months at 70° C. Also, it has been possible to 
devise quantitative neutralization tests based upon 
bioassay procedures for studying immunological 
relationships of this virus with other murine 
leukemia virus strains or isolates. 

Kinetic studies on the growth of virus in intact 
mice were also conducted by Dr. Rauscher, using 
quantitative bioassay procedures. Plasma or 
spleen removed from mice within 1 day after in- 
fection contained little or no virus, and virus could 
not be recovered through the third to fourth day 
after inoculation. Beginning on the fifth day virus 
was found to be present in both spleen and plasma 
and to increase with time at a logarithmic rate 
until a maximum was reached which persisted until 
the development of overt disease. No significant 
difference was found in the time to appearance of 
virus in spleen and plasma, but the quantity of 
virus reached higher levels in the plasma in some 
instances. 

Of significance also in indicating that this virus 
may differ entirely from other isolates are the 
pathogenesis studies of Dr. Dunn carried out in 
collaboration with Dr. Rauscher. Unlike the 
murine leukemias induced by the Gross and 
Moloney viruses, the thymus appears not to be the 
primary site of leukemogenesis or of viral 
synthesis. 

Dr. Fink has initiated serological investigations 
on the Rauscher strain virus and has succeeded in 
producing relatively potent antiserum in rabbits. 
Also, she has developed vaccines with formalin- 
inactivated and heat-inactivated virus, combined 
with Freund's adjuvant, which effectively pro- 
tected mice against challenge with strong doeses of 
virus. Vaccinated mice also showed high levels 
of neutralizing antibodies, as determined by Dr. 
Rauscher in bioassay studies. The knowledge that 
a strong immunity can be developed to another 



NATIONAL CANCER INSTITUTE 



51 



murine leukemia virus (Dr. Friend was also suc- 
cessful in vaccinating against her agent) increases 
the hope that if human leukemias are caused by 
viruses, some of them may be effectively controlled 
by classical immunization procedures. While 
immunological studies on the Rauscher strain 
virus are incomplete, preliminary findings indi- 
cate that there is no strong antigenic similarity 
between this strain and the Friend, Moloney, and 
Schoolman-Schwartz isolates. 

Dr. Zeigel, in collaboration with Dr. Rauscher, 
has studied the morphology of the Rauscher strain 
agent under the electron microscope in both tissue 
and blood using both the thin section and the nega- 
tive staining technique. Morphologically, the 
virus is indistinguishable from the Moloney strain 
agent, including the existence of a tail and an 
hexagonal body as revealed by negative staining. 
Up to 80% of the virus particules were found to 
have "tails." 

Studies by Dr. Manaker on the Rauscher strain 
virus showed that it, like the Moloney strain, could 
not be propagated in tissue cultures of human or 
monkey bone marrow, although it could be grown 
in cultures of mouse bone marrow. Nor could this 
virus be grown in mouse liver cell cultures. 

Dr. Manaker has continued to study the isolate 
of mouse leukemia virus which he reported last 
year. Serial blind passages of extracts prepared 
from mouse spleens and thymus glands were made 
14 days after neonatal virus inoculation, and sev- 
eral weeks before leukemia could be expected to de- 
velop. Sixteen successive passages were carried 
out. Five further passages were then made at 
10-day intervals. Some mice of each passage 
group were retained for observation of leukemia 
development. Leukemia has been observed 
through all passages, indicating that early virus 
proliferation occurs long before leukemia may be 
expected to develop. Other experiments were de- 
signed to determine how soon appreciable quanti- 
ties of virus appeared in mice following the initial 
virus inoculation. It was found that new virus 
was present within 10 days in mice that had been 
injected when newborn. When older, immuno- 
logically competent mice were inoculated, new 
virus was observed at 7, 21 and 28 days after inoc- 
ulation, but not at 14 days. The failure of at- 
tempts to recover virus at 14 days in the immuno- 
logically competent animals is thought to be due 



to the production of inhibitors (e.g., antibody), 
the subsequent reappearance of active virus being 
due to saturation of available inhibitor with the 
increasing production of virus. Further inves- 
tigations on this phenomenon are in progress. 
Other studies by Dr. Manaker on the effects of 
cortisone, and the enhancement of very low 
(subclinical) doses of virus are in progress, but 
the results are incomplete. These studies are di- 
rected toward the establishment of methods which 
might enhance the chances of recovering viral 
etiological agents from human leukemia, if such 
exist. 

Previous reports have described the finding of 
Dr. Zeigel of viral type particles budding from 
the membranes of acinar cells of the pancreas in 
apparently normal chicks and chick embryos. 
Similar particles were observed in other tissues of 
the same birds, but not in the process of budding. 
Birds which failed to show budding in the pan- 
creas also failed to show virus in other tissues. 
These findings suggested that the particles might 
represent lymphomatosis virus which is known to 
be endemic in most flocks of chickens and which 
is passed through the egg in some instances. Also, 
they suggested that the pancreas might be the most 
important primary site of viral replication during 
subclinical infection. 

These studies have been followed up by Dr. 
Zeigel in collaboration with Dr. Rauscher of this 
laboratory and Dr. Burmester of the Regional 
Poultry Research Laboratory (USDA), East 
Lansing, Michigan. The following evidence in- 
dicates that the particles observed by Dr. Zeigel 
are very probably the virus which causes lympho- 
matosis in chickens. Hens from several colonies 
were isolated and trap-nested for identifying eggs 
with dams. As previously shown by Burmester 
and by Rubin, dams which shed lymphomatosis 
virus have a high percentage of infected eggs (and 
embryos) as demonstrated by biological tests. 
Contrariwise, hens which do not shed virus and 
which show no antibody to the virus yield eggs 
free from biologically demonstrable lymphoma- 
tosis virus. The results of Dr. Zeigel and asso- 
ciates to date show varying percentages of embryo 
pancreases from infected hens which have budding 
virus particles, whereas embryos from hens which 
do not shed virus have failed thus far to show elec- 
tron microscopic evidence of particles in the pan- 



705--6S5 — 63- 



52 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



creas. Parallel biological studies on the same 
preparations examined by Dr. Zeigel are being 
conducted by Dr. Rauscher. 

The procurement of human cancerous tissues 
and the methods of approach used in attempts to 
demonstrate viral etiological agents in association 
with them were described in detail in the report of 
last year. An initial series of 10 experiments in- 
volving as many different human tumor specimens 
was described and preliminary evidence of the in- 
duction of tumors in mice injected neonatally with 
the microsome fraction of one of the human tumors 
was reported. These experiments have now been 
completed and all mice have come to autopsy. 
Only one of the 10 experiments (HT-8), namely 
that on which the preliminary report was made 
last year, showed significant differences between 
test and control groups in the studies involving 
newborn mice. The specimen employed in this 
experiment was gastric carcinoma tissue from an 
elderly man. 

The most striking finding was the occurrence in 
all 3 experimental groups of carcinoma of the 
kidney a tumor type that has not been observed 
spontaneously in the strain of mouse employed 
(BALB/c) and only very rarely among all strains 
of mice. The polyoma virus was ruled out by 
monitoring tests on all mothers that supplied new- 
born litters and by tests on mice developing tumors. 
It is emphasized that carcinomas of the kidney 
have not been observed as a result of the polyoma 
virus, although this agent does induce sarcomas of 
the kidney. In addition to the kidney tumors, 
cysts of the liver were observed in most of the mice 
bearing this lesion and four of them also had 
carcinomas of the acinar cells of the pancreas, an- 
other lesion not observed spontaneously. The kid- 
ney tumors appeared first and most frequently in 
mice that had received microsome material plus 
Freund's adjuvant (group c), but the group that 
received microsome suspension in buffer (group i) 
was only slightly behind. These two groups 
showed highly significant differences from control 
groups in time of appearance, frequency, and type 
of tumors. The group which received crude ex- 
tract of the gastric carcinoma developed only one 
kidney tumor, late, but two tumors of types that 
occurred also in control groups appeared slightly 
earlier. These results leave no doubt that some 
factor associated with the human cancerous ma- 



terial induced a significant tumor response in mice 
injected neonatally. 

On a basis of these findings a larger study of 
human cancerous materials using newborn mice 
has been initiated. The latter study has not been 
under way sufficiently long for significant results. 

The finding by electron microscopy of virus 
particles in the blood and bone marrow of leu- 
kemic mice, and the development of a practical 
approach to the study of the human disease based 
on the animal model system by Doctors Dalton and 
Moloney was reported last year. The observance 
of virus-like particles in 11 of 14 human leukemia 
specimens was also reported. An additional elec- 
tron microscope procured for speeding up the 
human studies has now been in operation for sev- 
eral months and Mrs. Mitchell has been trained in 
its operation. Dr. George Porter has also joined 
Doctors Dalton and Moloney in the human study 
and is taking responsibility for the broader survey. 
A total of 47 human leukemic cases has now been 
examined, 20 of which were found to contain par- 
ticles with characteristics similar to the particles 
associated with murine leukemia. The processing 
of specimens was carried out by Dr. Moloney who 
also has injected particle postive materials into 
newborn monkeys and mice. The oldest monkeys 
have been under observation for only 6 to 8 months, 
and no neoplastic reactions have yet been obtained. 
Among the various test groups of BALB/c mice 
that were injected neonatally with human leukemic 
materials, 8 out of 100 in one group have developed 
hemangioendotheliomas at the sites of inoculation, 
2 out of 50 in another have developed thymic 
lymphomas, and 1 in a third group of 25 has de- 
veloped a reticulum cell sarcoma, within periods of 
7 to 12 months following inoculation. No tumors 
have yet occurred among comparable numbers of 
control mice. Although still preliminary in na- 
ture, these findings appear to be comparable to the 
findings with the solid tumor (gastric carcinoma) 
described in the foregoing section, and they further 
justify the continued investigation of newborn 
mice as a potentially valuable indicator of tumor- 
inducing (or enhancing) factors in human 
neoplasms. 

Following his success in isolating infectious nu- 
cleic acid from murine leukemic tissues, Dr. Mo- 
loney is now applying the same techniques to 
human leukemic tissues. The nucleic acid frac- 



NATIONAL CANCER INSTITUTE 



53 



tions have been injected into newborn mice, and 
1 newborn monkey. Drs. Manaker and Stewart 
are also testing the nucleic acid fractions in tissue 
cultures of human cell lines. 

Dr. Sarah Stewart is now spending most of her 
effort and facilities in intensive efforts to propa- 
gate virus from human leukemias in tissue culture. 
Primary cultures of human fetal tissues such as 
thymus, spleen, liver and bone marrow, as well as 
calf embryo spleen are used as substrate. Leu- 
kemic materials used for inoculation include bone 
marrow, cells from the buffy coat, extracts and 
concentrate prepared from tissues obtained at au- 
topsy, and "microsome fractions" separated from 
blood by differential ultracentrifugation (sup- 
plied by Dr. Moloney) . Explants of human leu- 
kemic bone marrow and buffy coat cells are also 
made and fluids from them are used for animal 
inoculations and electron microscopic study. 
Twenty such specimens from human leukemic pa- 
tients (or autopsy specimens) have been success- 
fully established in tissue culture. Several have 
shown "suggestive" viruslike particles under the 
electron microscope, and one, involving buffy coat, 
has shown definite cytoplasmic particles similar 
to murine leukemia viruses. The electron micro- 
scopic studies are being made by Dr. David Fer- 
reira (a visiting scientist in the Cellular Biology 
Section) and Miss Valentine. 

In two instances embryonic calf spleen cultures 
that had been inoculated with bone marrow or 
blood pellet material from leukemia cases positive 
for virus-like particles (determined by Drs. Dal- 
ton and Moloney) showed a marked increase in 
rate of proliferation as compared with the con- 
trols. The fluids of the "infected" cultures be- 
came milky in appearance because of the massive 
cellular proliferation. TJninoculated cultures did 
not exhibit this effect. The same two "infected" 
cultures also showed a marked increase in motility 
in comparison with control cultures, as determined 
by time-lapse cinemophotography. The latter 
studies were in collaboration with Mr. Davenport, 
of the Pfizer Company in Maywood, New Jersey. 
If the phenomena of increased proliferation and 
increased motility are found by further study to 
be caused by a propagatable viral entity, it is pos- 
sible that they may be used in the future as diag- 
nostic criteria. Such criteria are badly needed for 
tumor viruses of the classical type since most of 
them do not produce CPE or other morphological 



changes that can be recognized in in vitro systems. 

Encouraging preliminary biological results have 
also been obtained by Dr. Stewart with the same 
two "infected" bovine spleen cultures which 
showed increased proliferation and motility. Four 
out of 35 hamsters inoculated neonatally with 
fluids from one of the cultures, and 5 out of 33 
with fluids from the other have developed reticu- 
lum cell sarcomas within 414 to 11 months. One 
such tumor was observed after 9 months, among 
75 hamsters inoculated with fluids from control 
cultures. Although reticulum cell sarcomas are 
known to occur spontaneously among older ham- 
sters with low frequency, the higher frequencies 
of such tumors, and their appearance as early as 
41/2 and 5 months in animals of the test groups 
suggest enhancement of the neoplasia as a result 
of the experimental processes described. This 
finding is comparable to the enhancement of tumor 
development in mice by human materials as de- 
scribed in preceding sections. 

A total of 19 monkeys have been injected when 
newborn with fluids from tissue cultures of human 
leukemia explants or human cell lines that had 
been inoculated with leukemic material. Six have 
died and 13 remain. The oldest have been under 
observation for about 7 months. No neoplasms 
have been observed to date. 

Dr. Manaker is also carrying out extensive in- 
vestigations on human leukemia using cultures of 
human and monkey tissues as substrates. Particu- 
lar emphasis is being placed upon the use of bone 
marrow from normal adults. Ribs removed at 
operation for heart surgery, at the Clinical Cen- 
ter, represent the primary source of human mar- 
row. Fetal bone marrow and other tissues are also 
used when available. Viable human leukemic bone 
marrow obtained by aspiration at the Clinical 
Center has been the chief leukemic material in- 
vestigated. Marrow from 45 leukemic cases has 
been put into tissue culture, both as primary ex- 
plants and by seeding onto other human and mon- 
key tissue cultures. The cultures are incubated 
for 3 weeks and then carried through one or more 
additional passages. Fluids from the cultures are 
stored at low temperature and used as facilities 
permit for animal inoculation and electron micro- 
scopic examination for virus particles. Only a 
few preparations have been examined thus far by 
electron microscopy and none have shown virus 
particles. Biological tests in newborn mice have 



54 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



not been in progress for sufficient time to yield 
significant results. 

In collaboration with Dr. John Fahey (Metabo- 
lism Service, NCI), Dr. Dalton has made electron 
microscopic studies on bone marrow aspirates 
from a series of cases of multiple myeloma and 
macroglobulinemia. Among the multiple mye- 
loma cases at least two each of those producing 
gamma globulins, B2A globulin and Bence-Jones 
protein were included. Six cases of macroglobu- 
linemia have been studied. Characteristic cells 
with details of ultrastructure indicating their ori- 
gin from plasma cells were found in all cases, but 
the degree of differentiation was least in the cells 
from patients producing Bence-Jones proteins. 
The finding of characteristic cells in all six cases 
of macroglobulinemia is of interest because it had 
not been possible with the light microscope to make 
this identification. 

Dr. Merwin has continued her studies on the in- 
duction of plasma cell tumors in BALB/c mice 
with diffusion chambers. She has found condi- 
tions which yield incidences of plasma tumors as 
high as 45 percent in mice with empty chambers, 
as compared with the previous highest incidence 
of 16 percent. The chief factor in increasing the 
incidence appeared to be the size of the chamber. 
The techniques for producing a relatively high 
percentage of plasma cell tumors now makes pos- 
sible further practical systematic studies on the 
etiology of this tumor. The time of residence of 
the plexiglas was also of importance since among 
51 mice in which the material was removed at 
periods up to 6 months, only 2 (4%) developed 
plasma cell tumors. Nine months or longer was 
required for inducing the higher incidences of 
plasma cell tumors. Work is being continued on 
the effects of different types of viable tumor and 
other tissues within intact chambers placed in the 



peritoneal cavity. As reported last year, a par- 
ticular transplantable sarcoma of BALB/c mice 
maintained by Dr. Merwin caused an increase in 
plasma cell tumors over the frequency of such 
tumors caused by blank chambers alone. Another 
transplantable mammary gland carcinoma, prob- 
ably containing Bittner agent induced a high per- 
cent of sarcomas in the peritoneal cavity, and when 
Sarcoma 37 was included within chambers, leu- 
kemia and reticulum cell sarcomas resulted. Dr. 
Merwin is continuing these studies with particular 
interest in determining whether the chamber tech- 
nique may be used to detect small amounts of vi- 
ruses associated with mouse neoplasms which 
might be below the threshold of detection by other 
methods now available. If successful this tech- 
nique will represent a major advance in technology 
of work with tumor viruses. 

Dr. Zeigel has launched an important study of 
the fine structure of normal and abnormal mon- 
key tissues. Little electron microscopic work has 
been done on this type of laboratory animal th^s 
far, and relatively little is known even regarding 
the normal structure of different cell types. With 
the growing interest and use of non-human pri- 
mates in tumor-virus and other cancer research, 
baseline studies of the type being pursued by Dr. 
Zeigel are essential. Studies on normal cells of 
other laboratory animals are also being carried 
out by Dr. Zeigel. An investigation on the normal 
chick pancreas has revealed occasional ciliary proc- 
esses associated with the apical surfaces of acinar 
cells, centroacinar cells and duct cells. It was not 
established whether every cell, or every acinar pos- 
sessed at least one cilium. The cilia are similar in 
their fine structural character, however, to those 
cilia described in loci suggesting a sensory rather 
than a motile function. 



NATIONAL HEART INSTITUTE 



INTRODUCTION 

The Heart Institute has undergone a number 
of organizational rearrangements within the last 
year. Most of these changes were formally in- 
stituted in the final few months of the year. The 
reports of the heads of laboratories which follow 
and constitute the body of this report reflect the 
new organizational pattern although most of the 
last year's work had been accomplished before 
these changes had been effected. 

The modifications in the Institute's organiza- 
tional structure were necessitated largely by Dr. 
C. B. Anfinsen's acceptance of a position else- 
where. Dr. Anfinsen had been head of the Labora- 
tory of Cellular Physiology and Metabolism since 
the organization of the intramural program of 
the National Heart Institute in 1950. His depar- 
ture is a great loss to his associates, his program 
and the National Institutes of Health. Neverthe- 
less, the Heart Institute can consider itself fortu- 
nate in the relatively minor dislocation which has 
ensued. The administrative and scientific autono- 
my which the sections of Dr. Anfinsen's laboratory 
had enjoyed has made easy their assumption of 
laboratory status under excellent scientific leader- 
ship. Dr. Anfinsen's own section under Dr. Kielley 
has become a part of the Laboratory of Biochemis- 
try. Other changes have been (1) the abolition 
of the Laboratory of the Chemistry of Natural 
Products and the establishment of part of it under 
Dr. Fales as the Section on Chemistry, Laboratory 
of Metabolism; (2) the organization of the Section 
on Molecular Disease under Dr. Fredrickson in 
the Laboratory of Metabolism ; and (3) the estab- 
lishment of the Section on Biochemical Genetics 
under Dr. Nirenberg in the Laboratory of Clinical 
Biochemistry. The Heart Institute is fortunate 
to have been able to offer the space and facilities 
to Dr. Nirenberg for the expansion of his program. 

The reports which follow are those of the Lab- 
oratory Chiefs. We believe that the quality of 
the work they describe will speak for itself. 



LABORATORY OF BIOCHEMISTRY 

Section on Enzymes 

The activities of the Section on Enzymes con- 
tinue to be concerned with detailed studies of 
diverse metabolic processes. The specific en- 
zymatic systems under investigations are regarded 
as model systems and have been selected because 
they offer unique opportunities to delineate some 
basic biochemical mechanisms of more general 
biological significance. Attention has been fo- 
cused on the characterization of individual reac- 
tions and metabolites involved in (1) the biosyn- 
thesis of fatty acids, (2) the dissimilation of 
heterocyclic compounds, (3) the activation and 
utilization of one carbon compounds, (4) the ac- 
tivation of molecular hydrogen, (5) the dissimila- 
tion of amino acids, (6) the metabolism of ethylene 
glycol, (7) transsulfuration, (8) permeation and 
intracellular concentration of purines, (9) thiol- 
alkyl transfer reactions, and (10) the intracellular 
regulation of fatty acid biosynthesis. 

Metabolism of Heterocyclic Compounds 

Nicotinic Acid Dissimilation. Last year we re- 
ported the isolation from soil of an anaerobic 
bacterium which is capable of fermenting nicotinic 
acid with the formation of stoichiometric amounts 
each of acetate, propionate, C0 2 , and NH 3 . 

Insight into the mechanism of nicotinic acid 
dissimilation by this organism has been obtained 
by studying in parallel experiments the decompo- 
sition by cell suspensions of substrates specifically 
labeled in individual carbon atoms. The isotope 
from nicotinic acid-2-C 14 is found equally dis- 
tributed in the methyl groups of acetate and of 
propionate; the isotope from nicotinic acid-5-C 14 
is found equally distributed between the carboxyl 
group of acetate and the alpha group of propio- 
nate; the isotope from either nicotinic acid-6-C 14 
or from nicotinic acid-7-C 14 is found equally in 
the C0 2 and in the carboxyl group of propionate. 
These results are consistent with the conclusion 

55 



56 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



that nicotonic acid is either converted (1) to a 
symmetrical six carbon compound, or (2) that it 
is converted to two three carbon derivatives which 
are in isotopic equilibrium with each other. The 
latter conclusion is supported by the observation 
that pyruvate and lactate are readily fermented 
by cell-suspensions of the organism to mixtures 
of propionate, acetate and C0 2 . Moreover, dur- 
ing the decomposition of nicotinic acid-7-C 14 in the 
presence of a large pool of pyruvate, the pyruvate 
becomes labeled with C 14 . Under the latter condi- 
tions several other intermediates accumulate in 
significant concentrations. Two of these have 
been isolated in highly purified crystalline form 
and have been positively identified as a-methylene 
glutarc acid and 6-oxo-l,4,5,6-tetrahydronicotinic 
acid. In addition, 6-hydroxy nicotinic acid was 
established as an earlier intermediate. These re- 
sults are consistent with the working hypothesis 
that nicotinic acid degration involves the follow- 
ing reaction sequence: nicotinic acid— »6-hydroxy- 
nicotinic acid— >a-methylene glutaric acid— Kt-keto, 
y-hydroxy, y-methyl glutaric acid-*2 pyruvate-^ 
+ C0 2 + propionate + acetate. 

It was further observed that this organism con- 
tains exceptionally high concentrations of vita- 
min B 12 coenzyme. Preliminary experiments with 
cell free extracts suggest that this coenzyme may 
be implicated in the over-all nicotinic acid fer- 
mentation. 

Riboflavin Degradation. In an effort to learn 
more about the biological dissimilation of hetero- 
cyclic compounds, studies on the bacterial degra- 
dation of riboflavin have continued. As reported 
earlier, a pseudomonad isolated by the soil en- 
richment procedure has been shown to degrade 
riboflavin to urea, oxamide and 3,4 dimethyl-6- 
carboxy-a-pyrone with the intermediary forma- 
tion of l-ribityl-2,3-diketo-l,2,3,4-tetrahydro-6, 
4-dimethyl-quinoxaline (compound I) and 3,4-di- 
methyl-2, 3-quinoxalinediol (compound II). 
Ribose has now been tentatively identified as the 
other product formed in the conversion of com- 
pound I to compound II. This conversion re- 
quires the presence of molecular oxygen. 

Fatty Acid Synthesis. As noted in last year's 
annual report, the first step in fatty acid synthesis 



involves a condensation of acetyl CoA with mal- 
onyl CoA to produce an acetoacetyl- enzyme com- 
plex and C0 2 . Previous studies in this laboratory 
using cell-free extracts of Clostridium hhoyveri as 
a source of enzymes showed that this reaction in- 
volves the participation of two enzyme fractions, 
one of which is readily inactivated by heat (frac- 
tion A) whereas the other is relatively stable to 
denaturation by heat (enzyme II). 

Since much higher concentrations of these en- 
zymes are present in Escherichia coli, extracts of 
this organism have been selected for additional 
studies. Further purification of the two enzyme 
fractions involved in the condensation reaction has 
been made. The heat labile fraction A has now 
been separated into at least two sub-fractions both 
of which are needed in addition to the heat stable 
enzyme II for catalysis of the condensation reac- 
tion. One of these fractions catalyzes thioltrans- 
acetylation reactions between acetyl CoA and free 
CoA or suitable analogues such as pantetheine. 
The other labile fraction designated as enzyme I 
contains an essential sulfhydryl group which is 
protected from inhibition by N-ethylmaleimide by 
prior incubation of the enzyme with any of sev- 
eral fatty acyl CoA substrates. 

The heat stable enzyme II has been purified 
nearly 200-fold from extracts of E. coli. From 
sedimentation analysis in a sucrose density gra- 
dient it was shown to be a small protein with a 
molecular weight of about 10,000. The role of en- 
zyme II as the receptor protein involved in the 
acetoacetylenzyme complex formation is indicated 
by the observation that incubation of C 14 -malonyl 
CoA and acetyl CoA with fraction A and enzyme 
II leads to the formation of a C 14 -labeled aceto- 
acetylprotein complex which upon subsequent 
purification by salt fractionation and DEAE-cel- 
lulose chromatography remains associated with 
enzyme II activity. Incubation of the isolated 
C 14 -labeled acetoacetylenzyme II complex with 
fraction A, malonyl CoA and TPNH results in the 
formation of C 14 -labeled long chain fatty acids 
which are labeled in the third or fourth carbon 
atoms from the methyl terminal end of the mole- 
cules. This result supports the conclusion that 
the acetoacetyl-enzyme II complex is a bonafide 
intermediate in the synthesis of long chain fatty 
acids. 



NATIONAL HEART INSTITUTE 



57 



The Regulation of Fatty Acid Biosynthesis 

Previous studies in this and other laboratories 
have shown that the stimulation by citric acid of 
fatty acid synthesis in animal enzyme prepara- 
tions is due to a specific action on the activity of 
acetyl CoA carboxylase which catalyzes the syn- 
thesis of malonyl CoA. In continuing studies 
with purified preparations of acetyl CoA car- 
boxylase derived from rat adipose tissue, it has 
now been established that the activation of this 
enzyme by citrate is associated with changes in 
the sedimentation behavior of the enzyme. Ultra- 
centrifugation in a sucrose density gradient re- 
veals that the untreated (inactive) enzyme has a 
sedimentation constant of 18.8 S whereas active 
enzyme obtained by prior incubation with citrate 
has a sedimentation constant of 43 S. Both the 
activation and increase in sedimentation constants 
are reversed when the citrate concentration is de- 
creased by dilution. These results suggest that 
citrate induced activation of the acetyl CoA car- 
boxylase involves the aggregation of two or more 
inactive monomeric units. In view of the relative 
specificity of citrate for aggregation and because 
of the freely reversible nature of the activation, 
this appears to provide an effective control mech- 
anism for regulation of malonyl CoA formation 
and indirectly of fatty acid biosyntheses as well. 

The exact mechanism by which citrate exerts its 
effect on aggregation is not known. The possibil- 
ity that it serves merely as a chelating agent ap- 
pears to be ruled out by the fact that other strong 
chelating compounds are ineffective. Moreover, 
the possibility that a metabolic derivative of 
citrate is the actual activating agent is made un- 
likely by the observation that fluorocitrate also 
activates the enzyme. 

One Carbon Metabolism 

Formate Activation. The incidental observation 
that some crystalline preparations of tetrahydro- 
folic acid formylase obtained from Clostridium 
cylindrosporum catalyze the formation of formyl 
hydroxymate in the presence of ATP, formate and 
hydroxylamine was mentioned in last year's an- 
nual report. This observation has provoked a 
more intensive study to determine if the formo- 
kinase activity is a manifestation of the tetrahy- 
drofolate formylase enzyme or if it is due to a 



contamination of the latter enzyme with another 
formate activating system. The latter possibility 
is correct and has led to the discovery that cell- 
free extracts of C. cylindrosporum contain two dis- 
tinct formokinases which are separable by protein 
fractionation procedures. Both enzymes catalyze 
the reaction : 

ATP + formate^ f ormyl-P + ADP ( 1 ) 

The reaction catalyzed by one of these enzymes, 
formokinase I, is apparently nonreversible; this 
enzyme is further characterized by having high 
substrate specificity ; it catalyzes the activation of 
formate only. The other enzyme, formokinase II, 
catalyzes the activation of both acetate and for- 
mate in freely reversible reactions. In the pres- 
ence of coenzyme A and a transacylase present in 
the bacterial extracts, the formyl-phosphate 
formed by either formokinase serves as a formyl 
donor for the synthesis of formyl CoA. 

f ormyl-phosphate + CoA^± f ormyl-Co A ( 2 ) 
+ orthophosphate 

From equilibrium measurements of this reaction 
the AF was calculated to be — 1200 calories. 

The coupling of reaction 1 and 2 provides a new 
mechanism for the enzymatic synthesis of formyl 
CoA. Previous studies in this laboratory have es- 
tablished that formyl CoA is produced also by 
thioalkyl transfer of the CoA moiety from acetyl 
CoA to formate. The presence in cell free extracts 
of enzymes capable of forming formyl CoA and 
formyl phosphate calls attention to the potential 
role of these substances as activated one carbon 
metabolites in intermediary metabolism. 

The Oxidation of Methylamine. In search of a 
biological material particularly well suited for in- 
vestigations of fundamental mechanisms involved 
in the metabolism of one carbon compounds, sev- 
eral aerobic organisms have been isolated from the 
soil that are capable of utilizing methylamine as 
the sole source of carbon and nitrogen for growth. 
One of these organisms tentatively identified as 
belonging to the genus Pseudomonas was selected 
for more intensive study. Although several sugars 
and other simple compounds such as lactate, glyc- 
erol and pyruvate support growth, methylamine is 
the only single carbon compound of several sub- 
stances tested that Avill serve as a carbon source 



58 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



for growth. Nevertheless, other one-carbon com- 
pounds including methanol, formaldehyde and 
formate are actively oxidized by washed resting 
cell suspensions of organisms which had been 
grown on methylamine. Formate has been iden- 
tified as an intermediate in the oxidation of meth- 
ylamine to C0 2 and NH 3 by resting cell suspen- 
sions. Further insight into the mechanism of 
methylamine oxidation is being sought in studies 
with cell free extracts of the organism. 

Carbon Dioxide Activation. The net synthesis 
of acetate through the condensation and reduction 
of two molecules of carbon dioxide is catalyzed by 
numerous heterotropic microorganisms. Since this 
synthesis constitutes a major metabolic activity 
of Clostridium thermoaceticum, this organism has 
been selected as a source of enzymes for studies on 
mechanisms of carbon dioxide activation and 
assimilation. Previous studies in this laboratory 
have shown that the fixation of C 14 2 into acetate 
by cell-free extracts of C. theromaceticum is stimu- 
lated by additions of pyruvate, ATP, CoA, DPN 
and an electron donor system such as isopropanol 
and alcohol dehydrogenase. The acetate formed 
in the presence of C 14 2 is labeled mainly in the 
carboxyl group but variable amounts of isotope are 
found also in the methyl carbon atom. In addi- 
tion to its incorporation into acetate, C 14 2 is 
readily incorporated into the carboxyl group of 
pyruvate, formate and succinate. The possibility 
that serine is an intermediate in the synthesis of 
acetate is made unlikely by the fact that large pools 
of glycine and serine do not influence the rate of 
C 14 2 incorporation into acetate. Participation of 
vitamin B 12 coenzyme in the overall reaction is 
suggested by the observation that intrinsic factor 
inhibits the incorporation of C 14 2 into acetate. 
An intermediary role of succinate is suggested by 
the fact that C 14 -labeled succinate is readily con- 
verted to C 14 -labeled acetate and this conversion 
is inhibited by intrinsic factor also. 

Methane Fermentation. Another approach to 
the general problem of one carbon metabolism is 
made possible as a result of the isolation of the 
anaerobic bacterium Methanosarcina oarkerii in 
pure culture. This organism derives all of its en- 
ergy and carbon for growth through the fermenta- 
tion of methanol or acetate to mixtures of methane 
and carbon dioxide. Originally isolated by Sch- 



nellen in 1947, the organism was subsequently lost. 
Because of its unusual metabolism it has long been 
recognized as an interesting medium for the study 
of one carbon metabolism, but all efforts to re-iso- 
late the organism in pure culture in this and other 
laboratories, have until now been unsuccessful. 
During the course of its isolation it was discovered 
that pyruvate also serves as an excellent substrate 
for growth of the organism. Conditions for the 
rapid growth of the organism in large scale cul- 
tures are now under investigation in order to pro- 
vide sufficient material for more detailed studies at 
the enzyme level. 

Metabolism of Amino Acids 

Lysine degradation. Studies on the anaerobic 
decomposition of Lysine by cell-free extracts of 
two strains of Clostridia (Clostridium, stricklandii 
and Clostridium (Ecuador strain)) have been 
continued. Previous studies in this laboratory 
have shown that lysine degradation by both or- 
ganisms leads to the formation of one mole each 
of acetate and butyrate and to two moles of NH 3 . 
The subsequent finding that orthophosphate and 
ADP are needed for lysine degradation by cell- 
free extracts has led to the discovery that lysine 
decomposition is obligately coupled with the es- 
terification of one mole of orthophosphate to form 
ATP. Correct formulation of the overall reac- 
tion is therefore : 

ly sine + Pi + ADP-» ATP + acetate + 
butyrate +2 NH 3 

In addition to DPN, CoA, and 6,8-dimercapto- 
octanoate (lipoic acid) previously implicated, it 
has been established that vitamin Bi 2 -Coenzyme, 
Mg ++ , Fe ++ , pyruvate and acetyl CoA are required 
also as co-factors in the over-all enzyme system. 

The requirement for vitamin B i2 coenzyme, sug- 
gested in earlier experiments by the partial stimu- 
lation of charcoal-treated extracts by addition of 
pure dimethylbenzimidazole cobamide, was more 
firmly established by the discovery that intrinsic 
factor inhibits lysine degradation by cell-free ex- 
tracts of both organisms ; the inhibition is reversed 
by the addition of cobamide coenzyme equivalent 
to the cobamide binding capacity of the intrinsic 
factor protein. 

The catalytic level of acetyl CoA required for 
the overall reaction can be supplied directly or 



NATIONAL HEART INSTITUTE 



59 



generated from added acetyl phosphate or added 
carbamyl phosphate and a catalytic amount of 
acetate together with free CoA. 

So far no intermediates in lysine degradation 
have been detected. Some possible intermediates 
including monoamino derivatives and hydroxy 
analogues of lysine are not attacked by cell-free 
extracts; they are therefore, not probably inter- 
mediates. On the other hand a highly specific 
deacylase catalyzing the hydrolysis of a-N-acetyl 
lysine is present in extracts of both organisms, 
suggesting the possibility that an a-N-acyl deriv- 
ative may be an intermediate. 

The separation of at least one of the enzymes 
involved in the over-all reaction appears possible 
from the discovery that some enzyme preparations 
of the Ecuador Clostridium exhibit an almost 
complete dependency on the addition of a very 
small amount of a colored protein fraction derived 
from C . Stichlandii. This protein, presumably of 
rather low molecular weight, can be separated 
from the bulk of other protein by chromatography 
on Sephadex-C-100. The possible identity of this 
enzyme with the recently described iron containing 
protein "ferredoxin" is under investigation. 

Continued study of this complicated enzyme 
system promises to yield information on several 
fundamental problems such as reductive deamina- 
tion, anerobic phosphorylation, electron transport 
and vitamin B 12 coenzyme action. 

Cystathionine Metabolism. In the past year 
attention has been focused on continuing studies 
of the mechanism of pyridoxal-P potentiation of 
enzymatic elimination and replacement reactions. 
Formally, transsulfuration (the transfer of sulfur 
between cysteine and homocysteine) offers, in 
Neurospora, a complete spectrum of these possible 
reactions, being mediated by four reactions con- 
sisting of /?- and y-replacement, and /?- and y-elim- 
ination. 

The synthesis of systathionine by /?- and y-re- 
placement by Neurospora enzymes has been investi- 
gated. Synthesis by /^-replacement (from serine 
and homocysteine) has been demonstrated for the 
first time in a microorganism. This reaction, char- 
acteristic of liver, appears to be absent in E. coli. 
Its presence in me-2 mutants indicates that it is 
not catalyzed by the same enzyme performing /?- 
elimination from cystathionine, as had been pro- 
posed in the past. It does, however, appear to be 



catalyzed by rather highly purified preparations 
of the enzyme catalyzing y-elimination from 
cystathionine. 

During the past year, a preliminary report by 
English workers has described the enzymatic syn- 
thesis of cystathionine by y-replacement in an 
E. coli mutant. With very sensative methods for 
detecting cystathionine synthesis from cysteine and 
homoserine, a search has been made for this reac- 
tion in cell-free extracts of Neurospora without 
success. It is not yet certain that a different path- 
way is involved in Neurospora, although 3 genetic 
loci are implicated in the process, in contrast to 2 
in E. coli. Neither has it been possible to confirm 
the presence of the reaction in extracts of yeast or 
wild-type E. coli. The search is now being con- 
tinued with Salmonella, in which the genetic re- 
sults appear to more nearly duplicate those in 
E. coli. The situation with regard to the two- 
systathionine cleavage reactions in Neurospora is 
somewhat changed since last year's report. Wild- 
type Neurospora appears to contain 2 separable 
cleavage enzymes, catalyzing predominately /?- or 
y-elimination from cystathionine. The former is 
absent from me-2 mutants, but the latter is not 
absent from me-7. A definite but elusive question 
remains as to the true separate identities of these 
enzymes since a number of conditions appear to 
bring about ft-y enzyme transformations. 

In last year's report we described studies which 
identified the mechanism of a /^-elimination (from 
L-cystine) catalyzed by a cystathionine y-cleavage 
enzyme purified from Neurospora. The same en- 
zyme has now been used to gain an unexpected 
insight into the mechanism of y-eliminations 
(from cystathionine or homoserine) catalyzed by 
the same enzyme. N-ethylmaleimide has been 
found to trap a highly unstable precursor of a-ke- 
tobutyrate in these reactions, though it does not 
interact with any comparable intermediate in 
^-elimination (from lanthionine) catalyzed by 
the same enzyme. The product of the reaction 
has not yet been identified, but present results sug- 
gest that it is an acylvinylglycine, N-(N-ethylmale- 
yl)-vinylglycine. The immediate significance of 
these results lies in the fact that intermediary 
formation of free vinylglycine would establish a 
unique reaction course for the y-elimination reac- 
tions, and would pinpoint the point of bifurcation 
in the reactions by which homoserine yields a-keto- 
butyrate, and P-homoserine yields threonine. 



60 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



This bifurcation point would lie in alternate pro- 
tonation of an a or a y carbon, and would be quite 
analogous in this respect to the divergence leading 
to either racemization or transamination. 

Thioalhyl Transfer 

Thiolester substitutions mediated through the 
enzymatic transfer of thioalkyl groups from thiol- 
esters to free carboxylic acids are key reactions 
in diverse metabolic pathways. Such thiolester 
interchange reactions, especially those involving 
the transfer of the coenzyme A-moiety from one 
acyl CoA derivative to form another, constitute 
important biochemical mechanisms for the trans- 
fer of chemical energy from one intermediate 
metabolite to another. 

In an effort to establish intimate details of the 
enzymic mechanism underlying this fundamental 
reaction, studies have been initiated to examine 
the enzyme "CoA-transferase" that catalyzes the 
reversible transfer of the CoA-moiety of acetyl 
CoA to butyrate to form butyryl CoA and acetate. 
Clostridium aminobutyricum was found to con- 
tain relatively high concentrations of the "CoA- 
transferase." Optimum conditions for preparing 
cell-free extracts of this organism have been deter- 
mined ; conditions for the measurement of "CoA- 
transferase" activity have been defined and proce- 
dures for the partial purification of the enzyme 
have been developed. 

When enzyme preparations of sufficient purity 
have been obtained, the mechanism of the CoA- 
transfer reaction will be examined with the aid of 
isotopically labeled compounds. 

Permeation and Intracellular Concentration of 
Purines 

A new study in this laboratory is directed to- 
ward elucidation of mechanisms underlying the 
transport and intracellular accumulation of pu- 
rines. Biochemical studies of cellular transport 
systems have been hampered by the difficulty of 
identifying the components of the transport sys- 
tem which are presumably located in the cyto- 
plasmic mebrane. For this reason, the transport 
system of Bacillus subtilus has been selected for 
study since the cytoplasmic membrane of this or- 
ganism is readily isolated from the non-membra- 
nous parts of the cell. It is hoped that such mem- 



brane preparations can be used to form an 
artificial phase boundary across which the trans- 
port, or flux, of purines can be studied apart from 
the non-membranous parts of the cell. 

Preliminary to the more exhaustive studies 
planned, methods have been developed for the iso- 
lation of cytoplasmic membranes of B. subtilus 
and procedures for measuring the intracellular ac- 
cumulation of purines by the organism have been 
established. Using, 8-azaquanine resistance as a 
means of selection, several mutant stains have 
been isolated following ultraviolet light irradia- 
tion and have been found to be deficient in their 
ability to transport guanine but not adenine. It is 
hoped that direct comparisons of transport be- 
havior and of enzymes derived from the cellular 
membrane fractions of the mutant and wild-type 
stains will provide significant data relevant to the 
mechanism of purine transport. 

Hydrogen Activation 

Previous studies in this laboratory have shown 
that cell-free extracts of Clostridium kluyveri 
catalyze the reduction of DPN by molecular hy- 
drogen. Partial resolution of the hydrogenase 
system responsible for this reaction has led to the 
discovery that hydrogen-linked DPN reduction 
requires the. presence of Fe ++ , FAD, and an uniden- 
tified thermal stable cofactor which is present in 
boiled extracts of the organism. Research on this 
problem has now been resumed; the ultimate ob- 
jective is to isolate and identify the thermal stable 
cofactor and explore its role in other biological 
transport systems. 

Optimum conditions for the preparation of cell- 
free extracts of C. kluyveri have been determined 
and a new manometric assay of hydrogenase activ- 
ity has been developed. The new method involves 
measurement of hydrogen uptake associated with 
the reduction of pyruvate to lactate when the hy- 
drogenase-dependent reduction of DPN is coupled 
with pyruvate reduction in the presence of added 
pyruvate and lactic dehydrogenase. This method 
is superior to that formerly employed since the 
nonhydrogenase dependent blank reaction is neg- 
ligible and it permits a continuous measurement 
of the time course of the reaction. The nonlinear 
time course of hydrogen uptake exhibited by most 
enzyme preparations has been shown to be due 



to the presence of ferric iron and can be overcome 
by preincubation of the enzyme with glutathio- 
nine. 

Ethylene Glycol Metabolism 

Studies on the conversion of ethylene glycol to 
a equimolar mixture of acetate and ethanol have 
been continued. The organism responsible for 
this fermentation has been shown to be a new spe- 
cies of Clostridium and has been named Clostri- 
dium glycolicum. Acetaldehyde has been identi- 
fied as an intermediate in glycol decomposition by 
cell-free extracts. Treatment of cell-free extracts 
with charcoal results in enzyme preparations that 
are markedly stimulated by additions of Fe ++ 
pyruvate, a-ketoglutarate, serine, threonine, glu- 
tamate and boiled extracts of the organism. Lack 
of stimulation of dimethylbenzimidazole cobam- 
ide and the lack of inhibition by purified intrinsic 
factor appear to rule out the involvement of vita- 
min Bi2-coenzyme derivatives. These properties 
indicate that the conversion of ethylene glycol to 
acetaldehyde in this organism involves a mecha- 
nism distinct from that discovered in Aerobacter 
aerogenes by Abeles. 

Based on growth yield data it has been con- 
cluded that the dissimilation of ethylene glycol 
to ethanol and acetate is associated with the pro- 
duction of at least 0.85 moles of ATP per mole of 
ethylene glycol fermented. This is considerably 
in excess of that expected if the sole energy yield- 
ing reaction involves a dismutation of the acetal- 
dehyde intermediate to ethanol and acetate. At- 
tention is drawn to the possibility that the 
conversion of ethylene glycol to acetaldehyde may, 
in this organism, be an energy yielding process. 

Section on Cellular Physiology 

The program of the Cellular Physiology Sec- 
tion has continued in its fundamental objectives of 
investigating the structural basis of the biochemi- 
cal activity of proteins and their functional rela- 
tionships in the integrated activity of cellular 
structures. 

The hypothesis that the three-dimensional struc- 
ture of proteins is determhied by their primary 
amino acid sequence has continued as a major 
stimulus of the research. This thesis, originally 



NATIONAL HEART INSTITUTE 



61 



formulated as a consequence of observations on re- 
generation of native enzyme in the cyclic reduc- 
tion and reoxidation of disulfide bridges in the 
enzyme ribonuclease, and subsequently fortified 
by similar reversible alterations in the three-di- 
mensional structure of other enzymes, has been 
illuminated by the initiation of systematic studies 
on environmental factors and structural modifica- 
tions influencing enzyme reactivation during the 
past year. 

This hypothesis has also found support in inves- 
tigations on the molecular structure of the fibrous 
proteins of the contractile mechanism of muscle, a 
program initiated some time ago and of growing 
emphasis in the research of the section. 

In addition to studies focused on the structure 
of protein molecules, the program of the section, 
as reported below, includes investigations on more 
biological aspects of biochemistry such as the bio- 
synthesis of proteins, the biochemistry and cy- 
tology of cell transport and the mechanism of fat 
transport. 

Structure of Ribonuclease 

Systematic study of the conditions for formation 
of native ribonuclease in the reoxidation of the 
reduced, inactive enzyme has demonstrated that 
the rate of reactivation is inversely proportional to 
the protein concentration, that the reactivation 
proceeds more rapidly at 24° C. than at 37° C. 
and is accelerated by increasing pH. Similar oxi- 
dation studies using reduced egg white lysozyme 
gave similar results for the influence of protein 
concentration on the rate of reactivation. How- 
ever, in the case of lysozyme reactivation proceeded 
more rapidly at 37° C. than at 24° C. 

While several differences were noted between 
the two proteins in the influence of environmental 
conditions on the rate of reactivation, the findings 
with both enzymes, coupled with observations on 
the reactivation of reduced polyalanyl trypsin, 
further substantiate the hypothesis that the ter- 
tiary structure of proteins is determined by their 
amino acid sequence and requires no additional 
genetic control. However, these kinetic experi- 
ments emphasize the fact that the formation of 
native enzyme from the reduced form during spon- 
taneous reoxidation is not in keeping with the 
physiological requirements of protein synthesis. 



62 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



If formation of disulfide bridges in these molecules 
is considered to be the last step in the biosynthesis 
of the enzymes, then this spontaneous oxidation 
process under optimal conditions is exceedingly 
slow in contrast to the apparent rate of complete 
sjmthesis of the enzyme in the pancreas — and the 
optimal conditions in themselves are not uniformly 
"physiological." As a consequence of these con- 
siderations, the possibility of enzymic reoxidation 
was investigated and it was discovered that a sys- 
tem could be prepared from rat liver consisting 
of a washed microsomalfraction and a heat stable, 
dialyzable component which together lead to ex- 
tremely fast reactivation of ribonuclease under 
conditions which result in virtually no reactiva- 
tion in the absence of this microsomal system — 
conditions more in keeping with physiological 
requirements. These results suggest that a system 
may exist in vivo for the catalysis of the conversion 
of newly synthesized polypeptide chains to the 
corresponding native proteins. 

Investigation of the role of side chain inter- 
actions on the reoxidation process has demon- 
strated that 8 of the 11 amino groups in the ribo- 
nuclease molecule may be modified by the intro- 
duction of alkyl or polypeptide chains without 
altering the activity of the enzyme or its capacity 
to undergo reversible reduction-reoxidation. 
Three of the amino groups are resistant to poly- 
alanylation and are presumed buried in the struc- 
ture. These residues have now been identified in 
the amino acid sequence, and it has been observed 
that if the alanylation reaction is carried out in bi- 
carbonate rather than phosphate buffer one of 
these three, identified as lysine #41 in the amino 
acid sequence, is also reactive. Since alanylation 
of the epsilon amino group of this lysine residue 
leads to a parallel loss of enzyme activity, it ap- 
pears that lysine #41 is critically involved in the 
active center of ribonuclease. 

It has long been believed that rupture of the 
disulfide bridges of ribonuclease leads to conver- 
sion of the native three-dimensional structure of 
the protein to a randomly coiled polypeptide 
chain. This conclusion was based primarily on 
optical rotatory changes. Observations during 
the past year on polarization of fluorescence meas- 
urements of ribonuclease indicate that the native 
molecule undergoes a subtle reversible structural 
transition above 36°C. — the temperature where 
it becomes susceptible to tryptic digestion and be- 



low the temperature at which optical rotatory and 
ultraviolet spectral changes are observed. Similar 
changes with reduced, carboxymethylated ribonu- 
clease are of interest in that they detect structural 
changes that may have significance in the mech- 
anism of regeneration of active euzyme in the re- 
oxidation process — that reduced ribonuclease 
may not be a random coil, strictly speaking, but 
may have residual structural elements that con- 
siderably restrict the possibilities in reformation 
of the four disulfide bridges in the molecule. 
However, this does not influence the genetic con- 
trol hypothesis mentioned earlier for it is known 
that reoxidation of reduced ribonuclease in strong 
urea solution, where all evidence indicates a ran- 
dom coil exists, leads to inactive enzyme which 
can, however, be transformed to active enzyme in 
normal aqueous solution by providing conditions 
for disulfide interchange. Under these conditions, 
all structural tendencies inherent in the amino 
acid sequence cooperate to produce the thermody- 
namically most stable form. 

It has been known for some time that limited 
digestion of ribonuclease with the proteolytic 
enzyme subtilisin leads to no loss of enzyme ac- 
tivity. However, when precipitated in acid solu- 
tion the enzyme separates into two parts RNase 
S protein and RNase S peptide, the latter repre- 
senting 20 amino acids from the amino terminal 
end of the molecule. These fragments, inactive 
by themselves, can be recombined to give a fully 
active enzyme. Since they are more susceptible 
to selective degradation by exopeptidases and en- 
dopeptidases, they offer an opportunity to study 
the essentiality of some portions of the structure 
for enzyme activity. At 25° C. RNase S protein 
treated with carboxypeptidase lost valine and 
serine from the C terminal end. The resulting 
product was active when combined with RNase S 
peptide but physical studies and observations on 
enzyme activity with respect to temperature indi- 
cated a lack of stability. If RNase S protein was 
digested with carboxypeptidase at 37° C. more 
extensive digestion was observed and the product 
was inactive in the presence of RNase S peptide 
and unable to combine with the latter judging 
from spectral studies. On the other hand, RNase 
S peptide was extensively digested by carboxy- 
peptidase converting it from a 20 to a 15 amino 
acid peptide without loss of ability to reactivate 
RNase S protein. Thus it appears that extensive 



NATIONAL HEART INSTITUTE 



63 



portions of the molecule, while essential for meet- 
ing the configurational requirements of the en- 
zyme, are fundamentally without influence on its 
activity. 

The ready formation of native ribonuclease 
from the reduced form has suggested the possi- 
bility of total synthesis with some chance of suc- 
cess. As an approach to this, experiments were 
designed for attacking the partial synthesis of 
the molecule. However, the chemistry of the 
process of recombination of fragments requires 
the blocking of all amino and carboxyl groups 
other than those involved in the desired reaction, 
and this blocking must be reversible. It has been 
found that the trifluoracetyl derivatives of amino 
groups and methyl esters of carboxyl groups can 
be readily formed and subsequently cleaved by 
treatment of the blocked enzyme with piperidine. 

Structure of Lysozyme 

Studies on the structure of egg white lysozyme 
have been completed. Techniques for peptide 
separation which have previously been perfected 
in this laboratory have been used to obtain a series 
of tryptic peptides from lysozyme. The sum of 
the amino acid compositions of these peptides is 
equal to the total composition of the enzyme, and 
thus the entire molecule can now be represented 
by this set of "subunits." Use of selective block- 
ing for sites of trypsin cleavage and use of other 
cleavage agents has yielded additional informa- 
tion which allows a tentative assignment of each 
"subunit" to a specific region of the enzyme. The 
characterization of the various peptide fragments 
of lysozyme has now been completed, and the ac- 
cumulated information makes possible the com- 
plete reconstruction of the amino acid sequence of 
this protein. 

Following short incubations of oviduct with ra- 
dioactive leucine, the newly synthesized lysozyme 
(containing the radioactive leucine) was isolated, 
and different leucine-containing peptides have 
been obtained by using techniques previously em- 
ployed in the amino acid sequence determination. 
Variations in the specific activity of the leucine, 
when compared with the peptide alignment for 
lysozyme, are consistent with a model which 
depicts protein biosynthesis as a process of unidi- 



rectional growth (along a special template) of a 
polypeptide chain beginning at the amino and 
terminating at the carboxyl end. 

Myosin Structure and Activity 

Studies on the structure and function of the 
fibrous proteins involved in muscular contraction 
are continuing. As a first approach to this prob- 
lem earlier reports presented successful efforts di- 
rected toward exact measurements of the molec- 
ular parameters of myosin. These efforts demon- 
strated that the long myosin molecule is composed 
of three polypeptide chains of identical structure 
wound together in the form of a three-stranded 
rope. This complex can be dissociated into single 
strands and reassociated by removal of the dis- 
sociating agent. Though enzyme activity has not 
been regenerated in these experiments, the results 
demonstrate the great tendency of this particular 
sequence of amino acids to form the fundamental 
a-helix structure in aqueous solution and to asso- 
ciate into a three-strandecl structure. The failure 
to recover enzyme activity appears to be due to 
disulfide bridge formation ; methods for prevent- 
ing the latter have not yet been devised. 

Other experiments demonstrated that a portion 
of the enzymically active center of myosin 
(ATPase) could be selectively blocked using ra- 
dioactive sulfhydryl reagents. Enzymatic diges- 
tion followed by peptide separations have pro- 
vided a means of isolating those portions of the 
molecule involved in the active center of myosin 
ATPase. During the past year methods have been 
developed for preparation of these fragments in 
pure form and in sufficient quantity for the deter- 
mination of structure. 

By very carefully controlled digestion of myo- 
sin by trypsin, it has been possible to isolate a 
portion of the myosin molecule, previously iden- 
tified as H-meromyosin, which retains the ATPase 
and actin-binding sites of the original molecule. 
The new procedure developed provides material 
devoid of internal fragmentation by trypsin. 
An earlier proposal placed this portion as a thick- 
ening of one end of the myosin molecule. Elec- 
tron microscope observations elsewhere have now 
verified the existence of these club shaped mole- 
cules. End-group analyses of our new H-mero- 



64 



Annual review of intramural research 



myosin lead to the tentative conclusion that the 
"club" represents the amino terminal ends of the 
polypeptide chains. 

Lipopeptides in Protein Synthesis 

Studies on the involvement of lipid compounds 
of amino acids in protein synthesis have been 
pursued further during the past year. A more 
extensive purification of some of the lipid amino 
acid compounds has been achieved. Some of 
these materials appear to be lipopeptides with 
specific fatty acids attached to the amino group 
of the peptide. Further structure studies indi- 
cate that the peptides are also bound in ester link- 
age to a polyglycerol phosphate. 

Studies initiated to examine more closely the in- 
volvement of the membrane structures of cells in 
protein biosynthesis have suggested on the one 
hand that a direct utilization of the energy avail- 
able from the electron transport network may be 
possible without the mediation of ATP. On the 
other hand, studies on the relationship between the 
nucleoprotein granules, ribosomes (known to be 
active in protein biosynthesis) , and the cell mem- 
branes suggest that the ribosomes attached to the 
membranes are more active than the free 
ribosomes. 

Cell Transport of Lipids 

Studies on the biochemistry and cytology of 
cell transport have proceeded along two main 
pathways. Investigations on the uptake of fatty 
acids and chylomicrons by lactating mammary 
gland have suggested an involvement of lipopro- 
tein lipase with the uptake of fat by this organ 
and support the hypothesis of involvement of this 
enzyme in the transport of triglycerides. The 
enzyme appears in the mammary gland only hours 
before parturition and disappears abruptly on 
cessation of suckling. Further studies on the 
lipid metabolism of mammary gland have dem- 
onstrated that this is one of the few tissues pos- 
sessing glycerol kinase and is thus able to synthe- 
size triglycerides from glycerol. 

In the course of studies designed to consider 
the function of cell membranes in cell transport 
using a mutant of the slime mold Dictyosteliwn 
discoidevm it became apparent that lipids of this 
organism are quite unusual. These lipids contain 
about 80% unsaturated fatty acids of which the 
two major ones are a previously undescribed di- 



unsaturated 16-carbon acid and a diunsaturated 
18-carbon acid. Studies with radioactive precur- 
sors have demonstrated the pattern of fatty acid 
synthesis. Myristate and palmitate are elongated 
to Ci 6 and C 18 fatty acids. The organisms have 
enzymes that then introduce double bonds spe- 
cifically and sequentially at positions 9-10 and 
5-6. 

LABORATORY OF CHEMICAL 
PHARMACOLOGY 

A. Mobilization and Utilization of Metabolic 
Substrates 

Function in the living organism is mediated 
through biochemical reactions; rapid adaptation 
to a changing environment is possible because cer- 
tain enzymes are activated by the action of the 
nervous system. All kinds of behavior require an 
increased utilization and consumption of meta- 
bolic fuel-free fatty acids (FFA) from adipose 
tissue and glucose from glycogen. It seems logi- 
cal that adjustment to these demands is mediated 
by the CNS since regulation of the supply of met- 
abolic fuel is as an integral a part of behavior as 
regulation of breathing and cardiac output. 

We have shown that adipose tissue contains 
much more norepinephrine (NE) than needed to 
control the circulation, suggesting that control of 
the output of FFA, as well as glucose, is a func- 
tion of the sympathetic nervous system. Last 
year we presented some evidence of this when we 
showed that stimulation of sympathetic fibers in- 
nervating adipose tissue in situ can increase the 
FFA in effluent blood. 

Evidence, of an absolute requirement for the 
sympathetic system in mobilization of metabolic 
substrates was obtained by showing that exposure 
of rats to 4° C, body temperature is maintained 
by production of heat through mobilization and 
burning of additional FFA and glucose. How- 
ever, if animals are p retreated with Ecolid (gan- 
glionic blocking agent) , they no longer can mobi- 
lize FFA or glucose, and they die in a few hours 
with a reduction of body temperature to 13° C. ; 
however, if they are also given epinephrine in oil, 
the rats maintain temperature and their ability to 
mobilize metabolic substrates. 

Evidence of the importance of the sympathetic 
system in mobilization of substrates was obtained 
using new technics of chemical sympathectomy; 



NATIONAL HEART INSTITUTE 



65 



rats are adrenal demedullated and then given 
either a nonsedative dose of reserpine or BW 
392C60, a potent bretylium-like compound which 
prevents release of NE by nerve impulses. On ex- 
posure of these animals to cold, FFA and glucose 
are not mobilized; the body temperature drops 
and the animals die at 15° C. in about 3 hours. 
However, pretreatment of rats with epinephrine 
in oil sustains body temperature and life. 

The sympathetic nervous system regulates out- 
put of FFA and glucose through catecholamine- 
induced activation of adipose tissue lipase ( ATL) 
and liver phosphorylase respectively. Thus, cold- 
exposure or the administration of catecholamines 
rapidly activates lipase and phosphorylase and in- 
creases FFA and glucose output. After chemical 
sympathectomy, cold-exposure no longer activates 
lipase and phosphorylase, or increases output of 
FFA and glucose. 

Although lipase is activated by epinephrine (E) 
as well as NE, studies with adrenal demedullated 
rats exposed to cold show that NE released at nerve 
endings is adequate to cause a maximal mobiliza- 
tion of FFA; but that E from adrenal glands is 
needed to mobilize adequate amounts of glucose. 

Chemical sympathectomy also prevents the mo- 
bilization of FFA and glucose in other situations. 
For example, sympathectomized rats do not ac- 
tivate ATL or increase the output of FFA (or 
glucose) after heavy muscular exericse, or after 
administration of a ganglionic stimulant, large 
doses of alcohol, morphine or depot ACTH. 

These studies have reoriented our thinking 
about the essential role of the sympathetic nervous 
system in energy-producing processes, for the 
chemically sympathectomized animals, exposed to 
cold, exercise and other stresses, exhibit the clas- 
sical shock-like response shown by adrenalec- 
tomized animals. 

In fact, adenalectomized and chemically sym- 
pathectomized rats show identical responses to 
stress such as cold-exposure ; in both types of ani- 
mals the ATL and liver phosphorylase are not 
activated; FFA and glucose are not mobilized; 
and the animals die in 3 to 4 hours at a body tem- 
perature of 15° C. Pretreatment of adrenalec- 
tomized rats with epinephrine in oil does not bene- 
fit these animals but if they are pretreated with 
corisone they can now activate lipase and mobilize 
metabolic substrates. If the adrenalectomized rats 
are pretreated with aldosterone they can also mo- 



bilize metabolic substrates. Apparently the effects 
of adrenalectomy on mobilization of substrates 
stem from failure of sympathetic receptors to react 
to catecholamines possibly because of an unfavor- 
able electrolyte environment. 

FFA Mobilization in Starved Rats 

Plasma FFA levels and adipose tissue lipase 
activity are markedly elevated in starved rats. 
These changes are not prevented by chemical sym- 
pathectomy, indicating that, in this circumstance, 
lipase in adipose tissue is activated by an entirely 
different mechanism from that involved in ordi- 
nary stress. 

B. Control of Basal Metabolism 

Treatment of mice with triiodothyronine dou- 
bles the 2 consumption. This effect is prevented 
by ganglionic blockade or by chemical sympathec- 
tomy, indicating the close interrelationship of the 
thyroid and the sympathetic system. Since the 
turnover of NE in peripheral tissues is unchanged 
by triiodothyronine it seems probable that one of 
the effects of the hormone is to sensitize the sym- 
pathetic receptors to catecholamines. 

C. The Neurochemical Transducer 

Adaptation, or response to environmental stim- 
uli, requires no new function but alterations in 
intensity of existing function and is mediated by 
changes in the level of hormones at reactive sites. 
We have introduced the term "neurochemical 
transducer" to describe those units at nerve end- 
ings that synthesize and store a neurohormone, 
and release it to receptor sites. (Biologically 
these may be considered as the basic units of 
behavior.) 

The following model now best represents the 
NE neurochemical transducer: NE is isolated 
from receptors and inactivating enzymes by a 
lipoid membrane and is present in at least two 
pools — a mobile, readily available pool, and a re- 
serve pool, presumably complexed with ATP in 
granules. NE in the mobile pool is sequestered 
within the membrane by active transport (a 
"pump and leak" system). Monoamine oxidase 
(MAO) outside the membrane metabolizes NE 
which diffuses out and ensures that, despite the 
continuous synthesis of amine, the steady-state 
level is maintained below that which saturates the 
transport system. The nerve impulse counteracts 



66 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the pump in front of the receptor, thereby releas- 
ing some NE. Between each nerve impulse, the 
pump is restored and since the receptor is ex- 
tremely close to the storage membrane, the NE 
released onto the receptor is pulled back into stor- 
age depot by the action of the pump. 

A picture of the NE neurochemical transducer 
is built on the basis of the following data : 

( 1 ) Uptake of C 14 NE by tissue slices shows that 
the amine is taken up against a concentration 
gradient by a process blocked by reserpine and 
metabolic inhibitors. 

(2) The scheme requires that NE be continu- 
ously formed irrespective of nervous activity. On 
injection of H 3 NE (i.v.) the label is rapidly taken 
up by various tissues. A plot of radioactivity 
against time shows that the H 3 NE in tissues first 
declines rapidly but within 12 hours declines ex- 
ponentially. The exponential decline is the classi- 
cal picture of a substrate store turning over at a 
constant rate. Complete blockade of sympathetic 
tone (ganglionic blockade) does not appreciably 
reduce the turnover rate. These results show that 
NE is formed in excess and is either lost by release 
onto receptors or by leakage onto MAO. 

(3) The turnover of H 3 NE in rat tissues is slow, 
with a half life of about 15 hours for heart and 
about 40 hours for skeletal muscle. The failure of 
nerve stimulation to deplete NE is explained by 
the economy of amine loss at nerve endings. Sym- 
pathetic stimuli are pulsatile so that most of the 
NE released to receptors is pulled back into stor- 
age between each nerve impulse. Thus, continu- 
ous synthesis together with economy of release 
explains why nerve endings are not depleted by 
nerve stimulation. 

(4) Preliminary results show that NE storage 
sites have a high specificity for H 3 NE and rapidly 
reject E. 

By regarding the synthesis, storage, physio- 
logical release, metabolism and the receptor as all 
parts of a single control unit a broad picture of 
drug action is permitted. Thus, the NE trans- 
ducer can be affected by drugs in 8 ways: (1) a 
drug (e.g., neosynephrine) may mimic NE, (2) in- 
directly mimic NE by releasing NE (amphet- 
amine), (3) block the action of NE (dibenamine), 

(4) block metabolism of NE (MAO inhibitor), 

(5) block synthesis of NE (dopa decarboxylase 
and dopamine-/3-oxidase inhibitors), (6) block 
storage (reserpine), (7) block release by nerve 



impulse (bretylium), (8) activate process by 
which nerve impulses release NE (guanethidine). 

We have demonstrated drugs that act in each of 
these ways; furthermore the effect of drugs has 
supplied us with considerable additional informa- 
tion about the transducer. 

(1-2) Drugs like amphetamine act peripherally 
by releasing NE onto receptors but centrally they 
stimulate receptors by a direct action. In small 
doses, amphetamine prevents depletion of NE by 
guanethidine. This finding fits the view that 
guanethidine is a specialized sympathomimetic 
agent acting "irreversibly" on the same process 
normally activated by amphetamine and by the 
nerve impulse. 

(3) Sympathetic blocking agents can have a 
highly selective action. For example, isopropyl- 
methoxamine (BW 61-43) is an agent which has 
no obvious effect in normal animals but prevents 
mobilization of FFA from adipose tissue and glu- 
cose from glycogen by catecholamines. 

(4) MAO inhibitors lower blood pressure be- 
cause they also act like bretylium — that is, they 
prevent the release of NE from nerve endings by 
nerve impulses. 

(5) Compounds that block synthesis of NE in 
vivo (dopamine oxidase inhibitors) do not neces- 
sarily lower the levels in tissues. This is further 
evidence that NE released at nerve endings is used 
over and over. 

(6) Reserpine depletes NE by blocking the NE 
pump. As a result the amine diffuses out onto 
MAO and thus leaves the tissues as the deaminated 
form. 

(7) Bretylium and a number of synthetic 
phenylmethylguanidines (especially BW 392C60) 
prevent nerve impulses from releasing NE at 
nerve endings. These compounds also prevent the 
release of NE induced by guanethidine and block 
the action of amphetamine. 

(8) Guanethidine and a number of synthetic 
phenylethylguanidines deplete NE by a mecha- 
nism which is blocked by bretylium and is differ- 
ent from that of reserpine. The decarboxylation 
products of a-methyl-DOPA and a-methyl-m-ty- 
rosine may also act like guanethidine. 

NE seems to be released by guanethidine from 
the mobile pool directly onto receptor sites rather 
than onto MAO and thus enters the blood stream 
largely in the undeaminated form. Further evi- 
dence that guanethidine is released from the mo- 



NATIONAL HEART INSTITUTE 



67 



bile pool is shown by the kinetics of release which 
are first order until about 40% is depleted and then 
zero order presumably as release from granules 
becomes the rate limiting step. Finally, adminis- 
tration of guanethidine to rat 20 hours after giv- 
ing H 3 NE decreases specific activity of NE in 
heart indicating that there is a pool of endogenous 
NE which has not mixed with the label. In con- 
trast, tyramine given at this time also releases NE 
but increases the specific activity of H 3 NE in 
heart. This seems plausible only if guanethidine 
releases NE from the "mobile pool" and tyramine 
releases NE directly from the granules contain- 
ing NE complexed with ATP. 

D. Drugs That Interfere With Biochemical Con- 
trol Mechanisms 

Pituitary -Adrenal System 

A misconception in endocrinology is that tran- 
quilizing agents like reserpine, chlorpromazine 
and morphine, protect animals from stress by 
blocking the output of ACTH. These compounds 
in sedative doses actually produce a persistent 
pituitary-adrenal response so that the animals can 
no longer respond further to a stressful stimulus. 
Moreover, in large repeated doses the drugs can 
reduce the content of ACTH in the pituitary to 
about 25% of normal. 

Lipid Transport 

Each day a large amount of FFA is mobilized 
from adipose tissue ; part is burned in tissues but 
most is re-formed in liver to TGL which is then 
secreted as a lipid protein complex and carried 
back to be restored hi adipose tissue. Despite the 
large turnover of lipid the normal TGL in rat 
liver is only about 5 mg./g. A fatty liver might 
be produced by drugs that increase mobilization of 
FFA, or increase formation and deposition of 
TGL, or block secretion of liver TGL. Thus, 
large doses of alcohol and morphine produce fatty 
liver by increasing FFA mobilization through in- 
creasing sympathetic tone. The glucocorticoids 
also secreted in response to these drugs enhance 
deposition of TGL in liver. Drugs like CC1 4 act 
mainly by blocking the secretion of TGL from 
liver. 



Factors Which Affect Duration of Drug Action 

Enzymatic Mechanisms of Drug Metabolism 

(a) Overwhelming evidence is now accumulat- 
ing that nitro reductase oxidizes TPNH in air and 
is therefore a component of TPNH oxidase 

(1) TPNH- oxidase + TPNH->reduced 
TPNH oxidase +TPN 

(2) reduced TPNH-oxidase + 2 — ^active 
hydroxyl donor 

(2B) anaerobically reduced TPNH oxidase 
and nitro compound— >TPNH-oxidase+ ni- 
troso compound. 

(b) Studies of the mechanism of drug oxidation 
in liver microsomes are hampered by difficulties of 
solubilization. The claim by other workers that 
aniline hydroxylase is solubilized by a snake 
venom or by a pancreatic lipase appears to be an 
artifact since after these procedures the hydroxyl- 
ase activitiy in supernatant appears to be due to 
suspended particles. 

Distribution of Drugs 

Two new ways by which drugs are reversibly 
bound to tissues have been disclosed : 

(a) Imipramine, and perhaps the phenothia- 
zines, are localized in various body tissues by re- 
versible attachment to phospholipids. 

(b) Guanethidine and perhaps other strong 
bases are not bound to plasma proteins but are 
strongly bound to various tissues especially heart 
in vivo. The extent of binding to heart is drasti- 
cally reduced on homogenization of the tissue. 
Preliminary results suggest that guanethidine is 
taken up in tissue slices by a process having cer- 
tain characteristics of active transport. 

Activators and Inhibitors of Drug Metabolism 

(a) In rats, methyltesetosterone and other ana- 
bolic agents induce drug enzymes. Phenobarbital 
and a large number of drugs, not anabolic agents, 
also induce drug enzymes. The two mechanisms 
have been shown to be different. 

(b) Liver microsomes are present in two forms : 
rough (containing the ribonucleic acid) and 
smooth-surfaced, and drug metabolizing enzymes 
are present mainly in latter. After induction of 
enzymes by phenobarbital or 3,4 benzpyrene the 



705-685—63- 



68 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



number of smooth microsomes is markedly in- 
creased. 

(c) A new phenomenon opposite to that of 
drug enzyme stimulation has been discovered. 
Morphine given to male rats in single doses de- 
presses the activity of the microsomal enzymes 
which dealkylate morphine, demethylate amino- 
pyrine and hydoxylate aniline and hexobarbital. 

(d) "Heat-labile" endogenous inhibitors of 
drug metabolism have been reported to be present 
in liver nuclei and microsomes. A major part of 
this activity is due to presence of pyrophospha- 
tases which destroy TPNH and TPN. 

Mechanism of Action of Desmethylimipramine, 
a New Antidepressant 

Previous studies have shown that imipramine 
(Tofranil) exerts its anti-depressant action 
through the formation of a metabolite — des- 
methylimipramine (DMI) a nonsedative, non- 
MAO-blocking agent. The action of DMI in the 
model system (reversal of effects of reserpine) is 
associated with the release of NE, thus: (1) Rats 
given DMI + reserpine show no excitation if ani- 
mals have been selectively depleted of brain NE 
by a-methyl-m-tyrosine. However, the combina- 
tion of drugs again produces excitation when the 
NE level has started to rise. (2) DMI produces 
excitation in rates only if given before reserpine, 
not if given after. (3) Further evidence that the 
action of DMI is related to catecholamines is the 
striking potentiation of DMI on the awaking ac- 
tion of DOPA given to reserpinized rats. 

Species Differences in DMI Metabolism 

Desmethylimipramine (DMI) shows an antide- 
pressant action in rats and man, preventing and 
reversing the action of reserpine, but has little ac- 
tivity in mice, rabbits and cats. In rats and man 
the drug has a long half life but in mice and rab- 
bits its half life is extremely short. In cats the 
half life of DMI is extremely long, but in cat brain 
NE is liberated by reserpine over a period of four 
or more hours compared to one hour in other spe- 
cies. This explains the failure of DMI to act in 
cats since we have shown in other studies that 
DMI acts only if NE is rapidly released. 



Passage of Substances Across Membranes 

(1) Membranes Within the CNS 

Studies of the distribution of drugs after mtra- 
cisternal or intraventricular injection are of value 
in identifying CNS compartments and in inter- 
preting drug effects after injection directly into 
the CNS. As further evidence that substances 
leave the CSF by a process of filtration, the rate 
of efflux of inulin was shown to be proportional to 
the pressure difference between CSF and dural 
sinus blood. However, a number of quaternary 
ammonium compounds including N 1 -methylnico- 
tinamide, hexamethonium and decamethonium 
leave by active transport. 

Incubation of various quaternary compounds 
with choroid plexus in vitro shows that they are 
taken up against a concentration gradient and 
that the process is depressed under anerobic con- 
ditions and by 2,4-dinitrophenol. 

The passage of lipid-insoluble drugs across the 
ependymal lining of the ventricles was found to 
be rapid. This means that in studying the effects 
of a lipid-insoluble drug in the CNS, the blood- 
brain barrier may be by-passed by injecting the 
drug directly into the brain ventricles. 

(2) Biliary Excretion of Drugs 

The mechanism by which the liver secretes qua- 
ternary ammonium compounds such as procaine 
amide ethobromide (PAEB) seems to be specific 
for strong ions since it does not act significantly 
on the tertiary amine derivatives. The process is 
saturable and is antagonized by other quaternary 
compounds provided they are also excreted in bile. 
The process is not depressed by organic acids such 
as bromsulphalein which are transported by an- 
other mechanism. 

For studies of bile secretion, an ideal substance 
should not be metabolized nor bound to protein 
or tissues and should be easy to assay. Such a sub- 
stance has been found in N-acetyl-PAH. 

{3) Penetration of Drugs Into Cells 

Certain quaternary ammonium compounds ap- 
pear to enter liver slices against a concentration 
gradient by a saturable transport process that is 



NATIONAL HEART INSTITUTE 



69 



blocked by anerobic conditions and by dinitro- 
phenol or iodoacetate. Similarly, the uptake of 
guanethidine by heart slices is blocked anero- 
bically and by dinitrophenol. 

(4-) Effect of Drugs on Uptake of Norepinephrine 
and 5HT 

A number of drugs interfere with uptake of 
NE and 5HT by various tissues. A number of 
these such as cocaine, chlorpromazine and imipra- 
mine are said to block the uptake of NE and 5HT 
but not to release them. Since desmethylimipra- 
mine (DMI) exerts no pharmacologic effects of 
its own, it provides a particularly useful tool for 
these studies. DMI does not change the rate of 
NE-C 14 uptake into red cells but markedly de- 
presses the active uptake of 5HT into platelets 
by competitive inhibition. However, after com- 
plete blockade of 5HT transport by N-ethylmalei- 
mide, DMI does not block the passive uptake of 



5HT. 



Unlike reserpine which completely blocks 5HT 
uptake, DMI only slows the rate of uptake. This 
may explain the failure of DMI to deplete endoge- 
nous NE. 

Other effects of DMI are: (1) it interferes with 
uptake of H 3 NE in vivo ; (2) blocks release of NE 
by guanethidine and tyramine but not by reserpine. 
These effects seem paradoxical and they do not ex- 
plain the drug's antireserpine action nor its ex- 
traordinary potentiation of administered NE. 

Development of New Drugs 

(1) Antidepressants 

Clinical trials show that DMI is active as an 
antidepressant in primary depression and acts more 
rapidly than the parent compound. In collabora- 
tion with Geigy, Switzerland, we are looking for 
the structural characteristics of active antidepres- 
sant DMI analogues. A number of analogues of 
DMI and of desmethylchlorpromazine have proved 
potent enough in preventing the action of reserpine 
to warrant clinical trial. 

(2) Dopamine Hydroxylase Blockers 
Collaborative studies with Smith and Nephew, 

England, have yielded structural characteristics 
for inhibitors of dopa decarboxylase and dopamine 
hydroxylase. Thus far we have found extremely 
active dopamine hydroxylase inhibitors but they 



have too short a half life in vivo to merit clinical 
trial. 

(3) Bretylium-Like Compounds 

In collaboration with Burroughs Wellcome we 
have shown that BW 392C60 ( N-o-chlorbenzyl- 
N',N"-dimethylguanidine) is the most potent 
bretylium-like compound yet found in antagoniz- 
ing the guanethidine-induced release of NE. This 
drug is now in clinical trial as a hypotensive agent. 
The greatest value of the compound is as a tool to 
produce chemical sympathectomy. Use of this 
compound has shown that completely sympathec- 
tomized rats cannot respond to a stressful stimulus. 

Development of New Methods of Analysis 

(1) Methods for analysis of dopamine and NE 
have been simplified and their sensitivity increased. 
(2) A new method for assay of FFA involving 
formation of lipid-soluble Cu ++ complex and assay 
of lipid-soluble copper has been applied to plasma ; 
results to date indicate that the problems of present 
titration methods may be obviated. (3) The level 
of NE hi tissues where the amounts are too low to 
be assayed by current methods can be assayed from 
the uptake of highly labeled H 3 NE tracer. (4) A 
sensitive coupling method to determine dopamine 
oxidase inhibitors has been developed. (5) 
Methods for determining whether enzymes are 
functionally blocked in vivo now include those for 
MAO, dopamine oxidase, and dopa decarboxylase. 

LABORATORY OF TECHNICAL 
DEVELOPMENT 

Gas Chromatography 

The major emphasis in the developmental part 
of this work continued to be on the development of 
methods of microanalysis using the techniques of 
gas-liquid and gas-solid chromatography. This 
included the development of methods of detection, 
methods for measurement of radioactivity in gas 
chromatographic effluents and procedures for ap- 
plying these techniques toward the solution of a 
variety of biochemical and physiological research 
problems. 

Five distinct but related methods for radioassay 
of labeled compounds analyzed by gas chroma- 
tography have been developed, each was explored 
and the usefulness and limitations of each defined. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Cumulative Collection on Anthracene 

The previously developed method of accumulat- 
ing the entire effluent in one detector filled with 
anthracene crystals while monitoring the radio- 
activity accumulated by scintillation counting was 
found to have the limitation that resolving power 
decreased during the course of the analysis. 

Fraction Collection on Anthracene 

The method of fractionation of the effluent into 
a number of separate anthracene filled vials for 
subsequent scintillation counting was developed 
and applied to a number of biochemical metabolic 
studies and evaluated. It became apparent that 
this technique offered the possibility of measuring- 
very small quantities of radioactive tracers with 
a resolution limited only by the frequency with 
which the effluent was fractionated. This method 
has proved useful in many studies conducted at 
NIH and has been adopted elsewhere as well. 

Ionization Chamber 

The method of measurement of radioactivity 
using an ionization chamber, started last year, was 
completed, evaluated, and the results published. 
The vapors in the effluent were combusted to car- 
bon dioxide and water, the water converted to hy- 
drogen gas and the gases led to a room temperature 
ionization chamber for measurement of radio- 
activity. 

Flow-through Scintillation 

A flow -through method for scintillation count- 
ing of Carbon-14 and tritium has been developed. 
A cartridge filled with anthracene crystals is used 
as a "flow-through" radiation detector. Upon 
leaving the column, the effluent is passed over hot 
copper oxide, which converts the organic materials 
to carbon dioxide and water. If tritium is to be 
counted, the water is reacted with hot iron to re- 
lease hydrogen and tritium gases. The effluent is 
then passed through the anthracene cartridge. 
The counting rate of the cartridge is monitored 
continuously during the analysis by a highly effi- 
cient, low-background, scintillation counter to 
yield a record of eluted radioactivity that re- 
sembles the conventional mass detector record. 

The detector is simply constructed, easily main- 
tained, insensitive to change in gas composition 
but highly sensitive to radiation. High efficiency 



(more than 70% for Carbon-14 and 20% for trit- 
ium) is coupled with low background counting 
rates (ca. 15 cpm at Carbon-14 settings, 50 cpm at 
tritium settings). The counting rate returns to 
this low background rate following the detection 
of a labeled compound. 

This method has been published, was used 
extensively in studies of the specificity of the 
esterification mechanism involved in fatty acid 
absorption in our laboratory, formed part of the 
methodology used in the "derivative ratio analysis" 
method for measurement of steroids, and was made 
available to many investigators at NIH. 

It possessed the advantage over the cumulative 
method of higher sensitivity without sacrifice of 
resolution, and the advantage over ionization 
chamber and geiger counters of insensitivity to 
change in gas composition. 

Tritium Assay 

Two methods of assaying tritium in compounds 
analyzed by gas chromatography were developed. 
One represented an improved method for convert- 
ing the tritium in the samples to tritium gas for 
assay by scintillation counter or ionization cham- 
ber, and the second a modification of the method of 
fractionating the effluent for subsequent radioassay. 
When the samples contain sufficient tritium in each 
component for accurate assay in less than 10 
seconds, the assay is performed during the analysis. 
The column effluent is passed through a combustion 
train consisting of : a heated tube containing copper 
oxide in which organic materials are converted to 
carbon dioxide and water; a second tube contain- 
ing heated iron, maintained in the reduced state 
by a stream of hydrogen gas, in which the water 
reacts to liberate tritium labeled hydrogen gas ; a 
magnesium perchlorate water trap ; and either an 
ionization chamber or a transparent tube filled 
with anthracene crystals for scintillation count- 
ing. When the samples contain insufficient tritium 
for radioassay during the analysis, the effluent is 
fractionated by being passed through a succession 
of cartridges containing p-terphenyl crystals 
coated with silicone oil. High boiling materials 
in the effluent are trapped and retained in these 
cartridges. Each cartridge is then transferred in 
its entirety to a vial containing DPO-toluene for 
radioassay by liquid scintillation counting for 
whatever time is required for statistically accurate 
results. 



NATIONAL HEART INSTITUTE 



71 



Determination of Blood Gases 

The micro-method for blood gas analysis was 
developed further with a new system for rapidly 
evolving the gases from the sample. The results 
agreed very well with Van Slyke determinations 
in the hands of the developer, but in the hands of 
a technician performing routine analysis the re- 
sults are not yet satisfactory. 

The D.G. Discharge Detector 

This detector has been shown to have very high 
sensitivity without the need for radioactive ion- 
izers, and is the basis for the micro-method for 
blood gases. Its high sensitivity and ability to 
measure atmospheric gases have led to its applica- 
tion in the gas chromatograph mounted in space 
and lunar exploration capsules. 

Quantitative Microdetermination of Lipids 

The effluent of a liquid-liquid or silicic acid 
chromatographic column may contain materials of 
interest dissolved in mixtures of solvents. Since 
the chemical nature of the solvents and solutes are 
often very similar, and since the composition of 
the solvents may be varied intentionally ("gradi- 
ent elution") during the chromatography detec- 
tion of the solute in solution may be difficult. 

The method is based on the difference in vola- 
tility between the lipid and the solvent. Aliquots 
of the solution are injected into a miniature gas 
chromatographic column operated at room tem- 
perature but containing only uncoated, solid re- 
fractory material. Carrier gas is passed through 
this column to a hydrogen flame ionization detec- 
tor. When the response of the detector indicates 
that the solvent has completely evaporated, the 
column is heated rapidly to 600° C, pyrolyzing 
the remaining material. The pyrolysis products 
are delivered to the flame ionization detector until 
the detector response again returns to baseline, at 
which time the heat to the column is turned off. 
The system is then ready for another injection. 

The electric charge carried during the passage 
of the pyrolysis products through the detector has 
been found to be a function of the total lipid 
injected. 

Application of Gas Chromatography Methods 

A method has been developed for microassay of 
complex biological molecules, such as sterols hav- 



ing specific functional groups. An unknown is 
acetylated with C 14 labeled acetic anhydride along 
with known material bearing the same functional 
group. When the two substances are separated by 
the gas chromatograph the ratio of the radio- 
activity is used to determine the ratio of standard 
to unknown. Sensitivity to nanograms of ma- 
terial is obtained. 

Collaborative studies utilizing the methods de- 
veloped were carried on in cooperation with other 
laboratories of NHI and other laboratories at 
NIH and elsewhere. Included in these are studies 
of the active agents in poison ivy for development 
of methods of standardizing the therapeutic ex- 
tracts, studies of the fatty acid composition of 
chyle and serum in relation to ingested fats, and 
studies of the part played by enzymatic activity 
in determining the lipid composition of chyle 
after ingestion of various fats. 

Ultra Microanalysis of Sodium and Potassium 

A method for measuring the content of so- 
dium and potassium in micropuncture samples 
obtained from renal tubules has been developed 
and applied. The method is based on the effec- 
tiveness of a helium plasma excited by a radio- 
frequency field to excite characteristic emission of 
Na and K. A sample of the order of several milli- 
microliters is suitably buffered with cesium and 
phosphate and pipetted onto a fine wire filament 
which is used to volatilize the sample into the 
glow. The characteristic emission is selected by 
an interference filter and measured with a photo- 
multiplier photometer. A peak reading voltmeter 
registers the response of the photometer which is 
linear over a range of 10" 11 to 10~ 12 moles of so- 
dium or potassium per sample. In application a 
standard error of ±2% has been obtained on 
single determinations. 

Ultra Microfreezing Point Depression Apparatus 

An apparatus for observing the thawing point 
of samples of biological fluids of the order of a 
few millimicroliters has been constructed. A 
thermoelectric refrigeration system consisting of 
a two stage Peltier effect thermocouple is con- 
trolled by a feedback servo that maintains the 
temperature of the samples at whatever level is 
set on the control dial. The rapidity of action, 
which is made possible by the electronic control 
and low thermal mass of the cold stage, allows 



72 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



one to control the rate of thawing so well that the 
point of disappearance of the last crystal can be 
approached rapidly — reversed and equilibrated 
under visual control. At present eight samples 
under oil in individual holes in a silver block are 
observed under a microscope. Samples and stand- 
ards can be interspersed in the same block to 
calibrate or provide interpolation points. 

Blood Flow Measurement 

The ultrasonic system for measurement of flow 
described in the previous report has been improved 
and somewhat simplified in a second model which 
utilizes sum frequencies for the intermediate fre- 
quency instead of requiring the method of multi- 
plication and conversion previously used. In addi- 
tion, a method of establishing the zero flow point 
electrically has been developed. This obviates the 
necessity of occluding the flow to obtain the zero 
flow calibration point. The high frequency 20-mc 
sound carrier used in this system makes it possible 
to utilize transducers of the order of 0.050" in 
diameter. A system for catheter tip flow measure- 
ment is currently being constructed. In perform- 
ance, the system has demonstrated superior fre- 
quency response and ability to resolve lower flows 
than other systems. Some spurious temperature 
sensitivity is also being investigated. 

Nuclear magnetic resonance techniques for flow 
measurements offer the unique advantage of being 
able to measure flow without mechanical or elec- 
trical contact with the blood vessel. It can, there- 
fore, measure total flow in an appendage or total 
number of protons in the sensitive region and can 
measure the passage of other resonant nuclei such 
as that of the fluorine atom. The apparatus has 
been tested for measuring continuous, pulsating 
and complex flow patterns, such as two coaxial 
flow streams in opposite direction or two separate 
tubes in the same sensitive area. Sensitivity suffi- 
cient to determine flows to reasonable accuracy in 
small vessels has been obtained by highly stabilized 
electronic circuits and determination of optimum 
sensor coil geometry. 

Fast Reaction Methods 

Fast reaction studies have added greatly to our 
basic knowledge of chemical and biological sys- 
tems. It was felt for some time, however, that in 
any reaction in which several steps were present, 
more than one detection method would add greatly 



to an understanding of these steps. The most 
obvious combinations are optical-thermal, optical- 
electron spin-resonance, plus adding electron con- 
ductivity to either of the two. Then thermo- 
dynamics and kinetics could be studied together. 
The final two combinations have been built. 

The work of the past year has gone mainly into 
greatly improving the optical-thermal apparatus 
for use in the study of the mechanism of the he- 
molysis of red blood cells. It will be extended to 
the detailed study of the model reactions of cata- 
lase, peroxidase, and other heme or hemocyanin 
reactions. A special flow apparatus has been built 
which mixes the reactants in 0.1 millisecond even 
when viscosities are as great as 1000 centipoise, 
and stops the flow in less than 8 milliseconds. 
About 2 ml. of solution are needed for experiment. 
The optical system will respond to changes of 
0.0001 optical density units even to 3.0 O.D. in less 
than 1 millisecond. The thermal system responds 
to 0.00002° C temperature changes in about 8 mil- 
liseconds. This is presently being reduced to less 
than one millisecond and a CoOp student from 
University of Cincinnati is improving the photo- 
multiplier tube to see 0.0001 O.D. or better in 0.1 
milliseconds, the latter improvements are neces- 
sary for the enzyme reactions. A 12-volt high- 
current tungsten light source stable to one part per 
million in intensity has been built as well as an 
all solid state temperature regulator which can 
detect less than 0.0001° C. temperature change. 

The special mixers that have been developed are 
of particular interest due to their fast mixing time 
and ability to handle solution of grossly different 
visocosities, i.e., water plus packed red blood cells 
(25,000 g's). 

Details of the mechanism of hemolysis have 
indicated that the cells may first go to hemolytic 
volume and then open allowing hemoglobin to 
diffuse out. Further work on this point is being 
done with the faster system. The earlier model 
also proved too slow to discriminate between the 
various steps in the carbonate reaction, and this 
is being repeated in the fast apparatus to explain 
the discrepancy between the optical and thermal 
data found in the literature. 

Calorimetry of Intact Cell Metabolism 

A differential calorimeter using 5 ml of solution 
and capable of measuring 0.00001° C. temperature 
changes has been built to follow the heat of reac- 



NATIONAL HEART INSTITUTE 



73 



tion of the sodium-potassium ATPase activity in 
human red blood cell membranes. A very high 
heat of absorption was observed, about 100-300 
Kcal/mole of P0 4 released, when the ATPase is 
poisoned by a cardiac aglycone such as strophan- 
thidin. Heat evolution appears to occur with the 
uninhibited system to the extent of 700-1000 
Kcal/mole of P0 4 released. Further work on this 
system may lead to an understanding of the mech- 
anism of this reaction. 

Photochemistry of the ATPase System 

The irradiation of the intact red blood cell 
ghosts with 366 m^ has shown that the free radical 
produced in riboflavin upon irradiation can react 
with the enzyme which hydrolyzes ATP when Mg 
is present and thus produce almost complete inhibi- 
tion. When Mg is removed and the irradiation 
carried out, the riboflavin radical is even more 
efficient than Mg in acting as an activator for the 
hydrolysis. In fact, while it is known that Mg 
alone will hydrolyze ATP without the enzyme, 
the process is very slow and the riboflavin radical 
acting without enzyme produces a reaction almost 
as fast as the enzymatic one. Preliminary trials 
using the enzyme alkaline phosphatase yielded 
similar results. A very crude trial to observe the 
free radical was carried out at Varian Associates 
with good results indicating a radical associated 
with the riboflavin and one associated with the 
enzyme. 

During the past few months a U.V. irradiator 
producing 10 19 photons per cm 2 second has been 
constructed and tested, riboflavin purified, the elec- 
tron spin resonance spectrometer operated and 
fiinally riboflavin irradiated in the cavity. Rather 
large drifts were produced in the spectrometer 
during irradiation and it is still unclear whether 
or not radicals were observed. Calibrating radi- 
cals indicated the machine was operating well, so 
that considerable work will need to be done to 
stabilize the system during irradiation. 

Similar work using gamma rays is being done in 
cooperation with the Greek Atomic Commission. 

Fluorescence and Phosphorescence 

Excitation of fluorescence and phosphorescence 
by electrons is well known and is highly efficient. 
Electron excitation of materials of biological inter- 
est was investigated by exposing about 50 lumines- 
cent of potentially luminescent materials to a beam 



of low energy electrons accelerated through a thin 
aluminum window directly into the sample. The 
low energy electrons were obtained from a simple 
electron gun provided with a pulsed high voltage 
system. Luminescence was measured by means of 
a photomultiplier photometer. In general, miner- 
als and crystalline scintillators and scintillators in 
toluene were effectively excited to luminesce but 
aqueous solutions and most fluorescent organic ma- 
terials were not much greater than the blank. The 
blank was shown to be in part due to luminescence 
of the nitrogen in the air and presumably could be 
reduced. The luminescence although not much 
greater than the blank would be useful if the blank 
could be eliminated by optical means. The appa- 
ratus has been rearranged to analyze the emission 
spectra of these materials and compare them with 
the fluorescence spectra. 

Measurement and Intracellular Localization of 
Tetracycline 

An assay method for the tetracycline antibiotics 
has been developed , based on fluorimetric measure- 
ment of the substituted anhydrotetracyclines 
formed when these antibiotics are heated in strong- 
ly acid media. This method has greater sensi- 
tivity and freedom from interference than the 
available photometric methods of analysis and 
greater simplicity, rapidity, and reproducibility 
than the existing fluorimetric method. For oxy- 
tetracycline it is also more sensitive than that 
method. 

Application of this method to the study of bind- 
ing of tetracycline to several tissues has shown that 
the ratio of drug concentration in the tissue to that 
in the suspending medium is about 3 for Earle's 
strain L cells in tissue culture, of the order 10 for 
isolated mitochondria from rat brain and liver, 
and 150-350 for E. coli taken as a typical, tetra- 
cycline-sensitive, Gram-negative rod. When the 
bound material, which is mostly bound reversibly, 
is washed out it appears both chemically and 
biologically to consist of unchanged tetracycline 
even after several hours exposure to the binding 
tissue. 

Experiments are partially completed to deter- 
mine the effect of various media and drug concen- 
trations on binding by bacteria and to explain the 
fluorescent microscopic observation that in living 
tissue culture cells intracellular bacteria are left 
unstained by the antibiotic at a concentration at 



74 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



which extracellular bacteria and intracecellular 
mitochondria are strongly stained. The impor- 
tance of the latter point to the question of carrier 
states following clinical use of tetracycline is 
apparent. 

Theoretical Analysis of Biological Transport 
Problems 

In the general analysis of input-output systems, 
it has been shown that the experimental assump- 
tions that are customarily made in carrying out 
any tracer experiment can be directly interpreted 
in terms of the axioms which characterize a linear 
vector space and a linear operator or mapping 
from one linear space to another. Hence, tracer 
input and output can be represented as vectors in 
a normed linear space and the relation of output 
to input is a continuous linear mapping of input 
space into output space. An immediate conse- 
quence is that if input space and output space are 
the same (i.e. the set of all continuous function of 
time) the set of all continuous linear transforma- 
tions is a normed operator algebra, when addition 
and multiplication are appropriately defined. 
Much of the analysis of particular problems can 
be carried out within this algebraic framework. 

Particular problems studied have been the time 
dependent counterflow problem and diffusion in 
non-homogeneous systems. An interesting result 
obtained in the counterflow problem is that for 
high membrane permeability the equation for the 
system reduces to 



h 



3 2 C 
5X 2 



dC 

5C 



where V is the velocity of flow and h the perme- 
ability. This is the diffusion equation. This equa- 
tion as one very interesting immediate result, 
namely that the diffusion coefficient depends on v2. 
This demonstrates that in a region supplied with 
blood by a counterflow system, small changes in 
velocity can make very large changes in washout 
time. 

LABORATORY OF CARDIOVASCULAR 
PHYSIOLOGY 

Studies on the Atrium 

The outcome of some of the studies done in this 
Laboratory in the past several years has been such 



as to indicate a substantial contribution of the 
atria to the regulation of the circulation. Two 
studies were completed this year which shed fur- 
ther light on this general subject. 

Atrial Fibrillation 

The question quite naturally arose as to what 
the effect of atrial fibrillation, per se, might be. A 
review of the literature did not uncover any satis- 
factory data. The lack of a satisfactory analysis 
of this question was attributable to the fact that 
when atrial fibrillation was induced ventricular 
irregularities also occurred and to the fact that 
reflex compensatory mechanisms, such as barocep- 
tor activity, tended to obscure the results. Accord- 
ingly, experiments were designed to circumvent 
these difficulties. Autonomic activity was abol- 
ished by vagotomy and ganglionic blocking agents. 
The ventricular irregularities were obviated by 
preliminary section of the bundle of His and then 
sequentially pacing the atrium and ventricle so 
that the normal temporal relationship between 
these atrial and ventricular excitation was re-estab- 
lished. In such a preparation when the atria were 
fibrillated, the hemodynamic consequences of the 
fibrillation alone could be assessed since neither 
ventricular irregularities nor reflex buffering oc- 
curred. Fibrillation was electrically induced and 
verified with bipolar electrograms. 

Several phenomena of interest were noted. 
When fibrillation was induced the atrial contribu- 
tion to ventricular filling was absent, mean left 
atrial pressure rose, left ventricular end-diastolic 
pressure fell as did aortic pressure and cardiac 
output. Forward flow fell by between 10 and 
20%. Analysis of pulse contours revealed the 
stigmata of mitral regurgitation to be present 
during atrial fibrillation, a finding not present in 
the control period. The presence of regurgitation 
during fibrillation was confirmed by the intraven- 
tricular injection of ascorbic acid with a record- 
ing platinum electrode in the atrium. 

These experiments may help in the evaluation 
of clinical atrial fibrillation with special regard to 
the question of why certain people with this phe- 
nomenon do well and others are obviously hurt by 
it. It seems clear that in the absence of other dis- 
ease, atrial fibrillation simply removes one of the 
contributory elements of a properly regulated cir- 
culation which is not, of itself, essential to an 
adequate (albeit a suboptimal) performance. 



NATIONAL HEART INSTITUTE 



75 



However, in the presence of other disease, atrial 
fibrillation can, by imposing an additional impedi- 
ment, produce a substantial increase in the symp- 
tomatology of circulatory failure. This comes 
into clearer focus if one examines the data from 
two other points of view. First, mean atrial 
pressure always had to be appreciably higher to 
produce a given hemodynamic stimulus to the for- 
ward propulsion of blood, i.e., left ventricular end- 
diastolic pressure. Further, the larger the ven- 
tricle and the stroke volume the greater was this 
difference. This does not occur with a normal 
sinus rhythm. Secondly, the pulse contours indi- 
cate that the same generality holds with regard to 
the volume of mitral regurgitation during atrial 
fibrillation, i.e., the larger the ventricle and the 
stroke volume, the larger was the mitral regur- 
gitant contour in the atrial pressure tracing. 

The Timing of Atrial Systole 

Further attempts were made to define the role 
of atrial function by use of the preparation de- 
scribed above. In this preparation the absence of 
atrial systole could be studied by turning off the 
atrial pace and observing the hemodynamic conse- 
quences of atrial asystole while pacing of the ven- 
tricle was continued at its previous rate. Further, 
the effect of changing the time of atrial systole 
could also be systematically studied. When atrial 
systole occurred in the range of 60 to 120 msec be- 
fore ventricular systole (AS-VS interval of 60 to 
120 msec), the atrial contribution to ventricular 
systole was effective and a mitral regurgitant 
wave was not seen in the atrium. When the 
AS-VS interval was longer than 120 msec, the 
mitral valve was apparently reopened by con- 
tinued inflow and a regurgitant atrial pressure 
pulse contour was observed with the subsequent 
ventricular systole. When the AS-VS interval 
was shorter than 60 msec a prominent pressure 
wave was observed in the atrium with ventricular 
systole but it is not possible to be certain whether 
this is the effect of continuing atrial systole alone 
or this effect plus mitral regurgitation. 

As with the studies on atrial fibrillation, the 
dispacement of atrial systole resulted in a higher 
mean left atrial pressure relative to any given 
left ventricular end-diastolic pressure. Both this 
gradient and the magnitude of the regurgitant 
pulse contour were greater when the heart and 
stroke volume were larger. With a properly 



placed atrial systole, ascorbic acid injected in the 
left ventricle did not appear in the left atrium. 
With an improperly placed atrial systole, the plat- 
inum electrode revealed clear evidence of mitral 
regurgitation. 

Mechanism of Closure of the Mitral Valve 

The two series of experiments described above 
strengthen the position arising out of previous 
work in this Laboratory indicating that the mitral 
valve can be preclosed (closed prior to the onset of 
ventricular systole) solely as a consequence of the 
activity of the atrium. This will certainly neces- 
sitate a reevaluation of certain phonocardio- 
graphic criteria which purport to use heart sounds 
as an indicator of the time of valve closure and 
also introduces the necessity for a reevaluation of 
the first heart sound. 

As far as the significance of these findings is 
concerned, they help to explain certain clinical 
observations such as the appearance of an early 
systolic murmur with atrial fibrillation when this 
occurs. Further, it now appears that an augmen- 
tation of atrial activity, such as occurs with sym- 
pathetic stimulation or during exercise, provides a 
mechanism as a result of which the total left ven- 
tricular stroke volume is propelled in a forward 
direction rather than allowing some of it to be 
devoted to mitral valve closure at the onset of 
ventricular systole. 

Studies on Homeometric Autoregulation 

The experiments which have been conducted in 
the last year relating to the phenomenon of ho- 
meometric autoregulation will be considered under 
two headings. One group of experiments had as 
its objective a further dynamic characterization 
of the phenomenon. A second group of experi- 
ments was aimed at a further examination of the 
possibility that th.Q compensatory response to an 
increase in activity is in some way related to the 
efflux of potassium that generally occurs when 
this phenomenon is observed. 

Characterization of the Dynamics of Homeo- 
metric Autoregulation 

The adaptive response of the heart to changes 
in rate was observed in experiments that were con- 
ducted in the areflexic dog, right heart bypass 
preparation. Heart rate was increased in a step- 
wise manner with mean aoritic pressure held con- 



76 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



stant. At each step, cardiac input and thus out- 
put were increased so as to hold stroke volume and 
stroke work constant over the entire range of heart 
rates studied. Quite remarkably, it was observed 
that left ventricular end-diastolic pressure re- 
mained essentially unchanged, i.e., the left ven- 
tricle (as a result of increasing stroke power) 
performed the same stroke work from the same 
end-diastolic pressure regardless of the rate and 
of the fact that a substantially shorter period of 
time was available to accomplish that work. 
Taken in its broadest meaning these data appear 
to indicate that when heart rate is increased at 
each rate the timing of the various phases of sys- 
tole, i.e., shortening of duration, are such as to 
compensate precisely for the altered time avail- 
able for each phase and thus produce the same 
stroke work from a given end-diastolic pressure. 
The existence of this rate-induced homeometric 
autoregulation makes unnecessary an invasion of 
the heterometric (Starling) type of autoregulation 
and the latter is thereby preserved for increases of 
stroke volume which cannot be accomplished in 
any other way (without the operation of external 
influences such as sympathetic stimulation) . 

Similar experiments were conducted to deter- 
mine the effect of increasing aortic pressure on the 
phases of ventricular systole. It was found that 
when aortic pressure was increased, as with an 
increasing heart rate, the heart adapted itself in 
such a manner that even though isovolumic systole 
was prolonged, the duration of ejection shortened 
by an equal amount so that total systole remained 
unchanged. As a result of this type of home- 
ometric autoregulation the heart pumping against 
a higher resistance performed much more stroke 
work, again without a change in left ventricular 
end-diastolic pressure, and thus held in reserve 
the heterometric (Starling) type of autoregula- 
tion for increases in stroke volume. 

It thus became possible to make two generalities 
about the performance characteristics of the heart. 
The first is that the heart is the kind of a pump 
which will, within limits, eject whatever is fed 
into it ; it accomplishes this by some presently un- 
known physicochemical phenomenon associated 
with an increase in fiber length (heterometric 
autoregulation). The second is that, whatever 
stroke volume is being ejected by it, the heart can 
continue to eject this volume against a wide range 
of resistances and over a wide range of heart rates 



without invading more than briefly the fiber length 
mechanism (homeometric autoregulation). An 
attempt is being made, as described below, to 
characterize aspects of the chemical changes asso- 
ciated with the latter type of phenomenon. 

Potassium Efflux During Homeometric Autoreg- 
ulation in the Metaoolically Supported Iso- 
lated Heart Preparation 

Potassium values in coronary arterial and 
venous blood were determined during pressure in- 
duced homeometric autoregulation and in some 
instances a net loss of K + was observed which ex- 
ceeded that observed after a large dose of acetyl 
strophanthidin. These K + losses were up to 2.5% 
of the total calculated intracellular K + . The 
amount of the K + loss appeared to be related to the 
increase in 2 consumption caused by the increased 
aortic pressure. Since left coronary blood flow 
was held relatively constant in such experiments, 
the A-V 2 widened and coronary venous blood 
content was lower. We therefore examined the 
possibility that a lower myocardial p0 2 might be 
the stimulus which promulgated the net K + efflux. 
This does not prove to be the case since, when we 
restricted coronary blood flow to a point where 
the A-V 2 was even wider and the coronary 
venous 2 content lower than during pressure- 
induced homeometricity, a net efflux of K + was not 
observed. The possibility was also examined that 
a particular type of effort the heart makes might 
be the stimulus for the net K + efflux independently 
of changes in 2 consumption. Accordingly, ex- 
periments were done in which, simultaneously, 
aortic pressure was lowered slightly and cardiac 
output increased greatly (work increasing four 
to five-fold) in such a way that 2 consumption 
remained unchanged. A net loss of of K* was not 
observed. It appears from the data obtained thus 
far that while a lowering of myocardial p0 2 is not 
the immediate cause of the loss of K + , something 
associated with the increase in the 2 consumption 
when this occurs as the result of increased activity, 
does stimulate the loss of this electrolyte. 

The hypothesis has been suggested that an in- 
crease in 2 consumption results in an increase 
in C0 2 production and that the consequent in- 
crease in intracellular pC0 2 or hydrogen ion con- 
centration in some way influenced the membrane 
of the cell so as to result in a K + loss. 



NATIONAL HEART INSTITUTE 



77 



Studies on Myocardial Metabolism 

Three types of study were done in this general 
area. The metabolically supported isolated heart 
preparation was used in each. The first was an 
examination of the influence of coronary blood 
flow on myocardial 2 consumption. The second 
was an examination of the effect of acetyl stro- 
phanthidin on myocardial 2 consumption; ob- 
servations were also made of the effect of this 
agent on the heart during myocardial hypoxia. 
The third not sufficiently advanced to merit more 
than mention here, was an examination of the 
influence of restricting coronary blood flow on 
lactate and pyruvate metabolism in the heart. 

The Influence of Changing Coronary Blood Flow 
on Myocardial 0. z Consumption 

Studies made elsewhere suggested the possibility 
that the amount of 2 consumed by the heart is 
in some way influenced by the amount supplied 
to it. The studies alluded to were of a type that 
did not make it possible to ascertain whether the 
activity of the heart remained constant when the 
coronary flow was varied. Since we knew from 
previous experience that a variation in certain 
aspects of the heart's activity could, of itself, 
cause a change in 2 consumption, it seemed to 
us worthwhile to examine this relationship while 
the heart's activity could be examined and held 
constant. Such an examination was possible in 
the isolated supported heart preparation. 

It is a reasonable assumption that if coronary 
flow is stopped, 2 consumption will cease soon 
thereafter. It is also reasonable to assume that 
above a certain level, even though one perfuses 
more blood through the myocardium, it will not 
use any more 2 . The matter of interest is, of 
course, what happens in the area in between. This 
has been determined. Varying coronary blood 
flow from about 100 to 600 ml per minute does not 
influence myocardial 2 consumption in this prep- 
aration. In the range just below 100 ml per min- 
ute a slight decrease in 2 consumption (about 
15%) can occur but this is always accompanied 
by evidence of deterioration of the heart's per- 
formance, i.e., rising left- ventricular end-diastolic 
pressure while heart rate, aortic pressure and 
stroke volume are held constant. If one attempts 
to reduce coronary flow any further a runaway 
rise of end-diastolic pressure occurs, mitral re- 



gurgitation appears and ventricular fibrillation 
will occur unless coronary blood flow is promptly 
augmented. 

In addition to the above, one interesting and 
unanticipated finding which appeared in each 
experiment was that, as coronary flow was gradu- 
ally diminished, left ventricular end-diastolic 
pressure rose prior to any decrease in 2 consump- 
tion. It, therefore, appears that the performance 
characteristics of the heart are influenced by the 
rate of supply (or washout) of some other sub- 
stance before the influence of myocardial hypoxia 
is felt. 

The Effect of Digitalis on Myocardial 2 Con- 
sumption 

Varying results have been reported concerning 
the influence of digitalis on myocardial 2 con- 
sumption and efficiency. It has been reported that 
when digitalis is given to normal man, myocardial 
efficiency falls. When given to the patient in con- 
gestive failure, myocardial efficiency rises. Such 
data have been used to support the position that 
digitalis acts differently in the normal heart than 
in the failing heart, We believe this to be an in- 
correct assumption and that closer examination 
of the meaningful relationships would reveal that 
the observed difference of the effect on efficiency is 
not based on any basic difference of the mode of 
action of the drug under the two sets of circum- 
stances. When digitalis was given to normal man, 
cardiac output fell, calculated work fell and effi- 
ciency therefore fell. In the patient with conges- 
tive failure, cardiac output rose, calculated work 
rose and efficiency therefore rose. What has been 
missing is an adequate appreciation of the ab- 
sence of any meaningful relationship between 
work and 2 consumption. The meaningful ex- 
periment is in our view, one in which the effect of 
the drug on 2 consumption is examined under 
circumstances wherein the activity and the type of 
activity of the heart is held constant. This has 
been determined. With the activity held constant, 
doses of acetyl strophanthidin which produced a 
marked increase in contractility had no discernible 
effect on myocardial 2 consumption. 

Remarkably, no data seem to be available on 
the question of whether digitalis increases con- 
tractility in the severly hypoxic heart. Accord- 
ingly isolated supported hearts were rendered hy- 
poxic by restrictions of coronary blood flow severe 



78 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



enough to produce marked elevations of left ven- 
tricular end-diastolic pressure. The administra- 
tion of acetyl strophanthidin under these circum- 
stances was followed by marked improvement in 
contractility without any change in myocardial 2 
consumption. 

Studies on the Synchronicity of Ventricular 
Contraction 

Previous experiments from this Laboratory 
demonstrated that changes in the pathway of ac- 
tivation and the consequent change in the syn- 
chronicity of ventricular contraction could modify 
the external work produced from any given end- 
diastolic pressure. This was shown by contrasting 
the ventricular systole observed during atrial pac- 
ing with that obtained during ventricular pacing. 
There is also a profound augmentation of ven- 
tricular systole during cardiac sympathetic nerve 
stimulation. The juxtaposition of these two find- 
ings gave rise to a potentially important question, 
namely, does cardiac sympathetic nerve stimula- 
tion achieve a portion of the augmentation effect 
by altering the pattern of myocardial activation in 
addition to the effect of the norepinephrine which 
is known to be liberated under these circum- 
stances ? 

Accordingly, studies were initiated to determine 
if, as a result of cardiac sympathetic nerve stimu- 
lation, the pathway or time required for ventricu- 
lar activation is altered in such a way as to have 
meaningful mechanical sequellae. Experiments 
were designed to measure conduction time through 
the A-V node, conduction velocity in the right 
bundle branch, surface activation of the left ven- 
tricle and also to estimate total ventricular activa- 
tion time. For these purposes bipolar electrodes 
were implanted on the atrium, bundle of His, right 
purkinje papillary muscle junction and on the 
surface of the ventricle at multiple points. Con- 
duction time between atrium and His bundle (an 
index of A-V nodal delay) is shortened by 
50-60% as a result of sympathetic nerve stimula- 
tion. Conduction time between His bundle and 
right purkinje spike is either unchanged or short- 
ened by less than 3% as a result of sympathetic 
stimulation. The pattern of surface activation of 
the left ventricle is not modified by sympathetic 
stimulation. The interval between activation of 
the right anterior papillary muscle and the base 



of the interventricular septum (an estimate of 
total ventricular activation time) is reduced by 
5-10% as a result of sympathetic stimulation. 
These data are taken to indicate that sympathetic 
nerve stimulation reduces the time for ventricular 
activation 5-10% by a mechanism apparently not 
related to its effects on the major distributions of 
the specialized conduction system and therefore by 
exclusion either in peripheral purkinje tissue, pur- 
kinje muscle junction or muscle. 

Studies on Reflexes Arising From the Heart 

In this general area attention was focused on 
the question of why, when a patient sustains a 
coronary infarction and hypotension results, there 
is not a greater and more consistent increase of 
peripheral vascular resistance. Experiments were 
conducted in which a hind limb was perfused 
through its isolated vessels at constant flow while 
changes in pressure were measured as an indicator 
of changes in resistance. With occlusion of the 
left anterior descending coronary artery or by in- 
terruption of blood flow to the left main coronary 
artery, neuronally mediated vasodilation was fre- 
quently seen even though carotid sinus pressure 
fell. This response was abolished by the interrup- 
tion of impulses in the vagi (either section or cool- 
ing) but not by atropine, thus establishing that 
the vagi carry the afferent limb of this reflex. 
Confirmation of the fact that the efferent limb of 
the reflex consists of a dimunition of sympathetic 
activity was obtained by the recording of electro- 
neurograms of the inferior cardiac nerve of the 
cat before and during occlusion of a coronary ar- 
tery. The impulse traffic was observed to diminish 
in spite of a lower arterial (and baroceptor) 
pressure. 

This work received recognition in the form of 
the Young Investigator's Award of the American 
College of Cardiology. 

Studies on Renal Function 

Extrinsic {Autonomic) Factors 

As described in detail in earlier reports, the 
studies in this laboratory concerning various fac- 
tors which modify renal function were initiated 
when it was observed that stimulation of the stel- 
late ganglion of the dog produced a striking di- 
uresis. Since the results of these studies indicated 



NATIONAL HEART INSTITUTE 



79 



that the diuresis was reflex in nature and possibly 
the result of stimulation of arterial baroreceptors 
by an increase in arterial pulse pressure, further 
studies were undertaken to determine the effects of 
changing arterial pulse pressure by carotid artery 
occlusion. In the early carotid occlusion studies 
renal blood flow (PAH), glomerular filtration 
rate (inulin), and electrolyte excretion did not 
show consistent changes during carotid occlusion. 
A perfused kidney preparation was therefore 
developed in which renal blood flow could be 
controlled and monitored. 

The specific details of the stellate ganglion stim- 
ulation experiments have been discussed in earlier 
reports. It appears that infusion diuresis may be 
mediated at least in part by the same mechanism 
as stellate stimulation diuresis, i.e., by withdrawal 
of renal vasoconstrictor tone. 

With the perfused kidney preparation it was 
possible to analyze those factors which contribute 
to the net response of the kidney to carotid occlu- 
sion. Using this preparation it has been estab- 
lished that carotid occlusion is always associated 
with an increase in renal vascular resistance, a 
change which can be substantially diminished by 
the intrarenal injection of an adrenergic blocking 
agent. This increase in resistance is observed 
whether or not renal blood flow is controlled by a 
pump. At the same time, it is well known that 
urine flow increases when renal arterial pressure 
is increased, independently of a change in renal 
blood flow or a change in renal vasoconstrictor 
nerve activity. Consequently, the rise in arterial 
pressure associated with carotid occlusion can con- 
tribute directly to the response of the kidney dur- 
ing occlusion. Therefore, the variability of the 
changes in water and electrolyte excretion by the 
kidney during carotid occlusion may represent a 
varying contribution of direct and reflex mecha- 
nisms to the total response. Thus, during carotid 
occlusion it is possible that total renal blood flow 
can decrease and urine flow and free water clear- 
ance increase with no change in the level of circu- 
lating antidiuretic hormone simply as a result of 
an increase in medullary blood flow subsequent to 
the increase in renal arterial perfusion pressure. 

The results of the stellate stimulation and caro- 
tid occlusion experiments, when put in proper pro- 
spective indicate that reflectes emanating from 
arterial baroreceptors represent a potentially im- 



portant mechanism whereby body salt and water 
homeostasis can be achieved. 

Intrinsic Factors {Autoregulation) 

In the course of these studies, the pressure-flow 
characteristics of the preparation could be ascer- 
tained. At the same time, it was of interest to de- 
termine those factors which may or may not con- 
tribute to the pressure flow relationships. As 
many have found previously, renal resistance was 
observed to increase as renal perfusion pressure 
was increased, i.e., the preparation autoregulates. 
Of interest, however, was the finding that little 
change in needle pressure (tissue pressure) is ob- 
served during autoregulation ; sudden maintained 
increases (70-90 mmHg) in ureteral and needle 
pressure produced a transient decline in renal 
blood flow and then a subsequent rise to near the 
control level indicating that the renal circulation 
autoregulates in spite of, rather than because of, 
an increase in tissue pressure. In contrast to find- 
ings of previous workers it was found that the de- 
capsulated kidney also does autoregulate. These 
experiments therefore show, we believe, that a 
change in tissue pressure or the presence of the 
renal capsule is not essential for the phenomenon 
of autoregulation. 

Renal Blood Flow Distribution 

It is generally accepted that renal medullary 
blood flow is only a few percent of total renal blood 
flow and that the medullary circulation does not 
autoregulate, the latter observation presumably 
accounting for pressure diuresis. At the same 
time it has been shown that the innervation of the 
renal medulla is scarce compared to the renal cor- 
tex. Experiments were initiated to determine if 
significant changes in renal blood flow distribution 
take place during such situations as changing 
renal nerve discharge, increasing renal perfusion 
pressure, etc. The transit time of cold saline is 
presently being used to estimate blood flow distri- 
bution changes. Although the experiments are in 
a very early stage, the results to date indicate that 
such transit times are much longer in the deep 
portions of the kidney than in the superficial por- 
tions. It is hoped that this approach combined 
with the perfused kidney preparation will provide 
a means whereby greater insight can be achieved 
with respect to those factors modifying both total 
renal blood flow and renal blood flow distribution. 



80 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Kallidin and Renal Function 

Over the past several years, studies from this 
laboratory have contributed significantly to the 
purification and identification of the hypotensive 
proteinases, called Kallikrein and the polypeptides 
kallidin. Partially as a result of these studies 
synthetic polypeptides are now available. Cur- 
rent evidence suggests that these polypeptides may 
be concerned with local blood flow regulation 
and/or distribution. Since the kidney excretes 
large amounts of both the enzymes, the polypep- 
tide studies have been undertaken to determine 
their effect upon renal hemodynamics as well as 
ivpon renal water and electrolyte excretion. When 
infused directly into the renal artery of the dog, 
kallidin produces an increase in renal blood flow 
and a striking diuresis and natriuresis, the latter 
characterized by both the increase in urine flow 
and an increase in urinary sodium concentration. 
Only a modest kaluresis occurs since urinary potas- 
sium concentration decreases. These experiments 
suggest that kallidin increases renal blood flow, 
that it may have a direct effect on electrolyte ex- 
cretion and thus represent another endogenous 
mechanism which contributes to salt and water 
homeostasis. 

Studies on Cathechol Amines 

Norepinephrine Refractoriness 

The role of catechol amines in circulatory regu- 
lation has been of interest to this laboratory for a 
period of years. The potent influences exerted on 
the myocardium have been discussed in detail in 
previous reports. Although there is little doubt 
that catechol amines exert a beneficial effect upon 
the myocardium it has also been established that 
when an animal is maintained on a constant in- 
fusion of norepinephrine, refractoriness develops, 
the mechanism of this is not yet known. It has 
been established that tissues can store large 
amounts of catechol amines and it has been sug- 
gested that the responsiveness to injected catechol 
amines may be diminished as a result of high tis- 
sue catechol amine content. Experiments were 
undertaken to determine whether agents which de- 
crease tissue catechol amine concentration can re- 
verse the refractoriness observed on constant 
norepinephrine infusions. The results to date in- 
dicate that ephedrine sulphate, a drug believed to 



release catechol amines from tissue can reverse 
norepinephrine refractoriness. This reversal can 
take place at a time when there is no significant 
change in arterial pH or arterial plasma catechol 
amine levels. 

Mechanism of Action of Ephedrine Sulfate 

During the above studies it was of interest that 
although ephedrine could increase arterial blood 
pressure substantially (100 mm Hg), there might 
be little or no change in arterial catechol amine 
levels. On the other hand, tyramine produces ap- 
proximately the same increase in pressure but a 
significant increase in arterial catechol amine lev- 
els. More detailed studies were therefore under- 
taken to determine the mechanism of action of 
ephedrine. The results of this study indicate that 
the extent to which ephedrine releases catechol 
amines is small, even in circumstances in which 
the catechol amine storage has been increased by 
catechol amine infusions. Thus it appears that al- 
though part of the effect of ephedrine is to de- 
crease tissue catechol amines, this effect alone can 
not explain the potent pressor responses observed. 

LABORATORY OF KIDNEY AND ELECTRO- 
LYTE METABOLISM 

The Laboratory of Kidney and Electrolyte 
Metabolism is pursuing a wide variety of activ- 
ities. These may be subdivided into four major 
areas which will be discussed separately. 

I. Renal Physiology 

a. Micropuncture Studies in the Dog 

In the last report, the development of a micro- 
puncture technique for the study of renal tubular 
function in the intact dog was described. In the 
past year considerable progress has been made. It 
has now been established with certainty that fluid 
throughout the accessible portion of the proximal 
nephron (the first 60%) is isotonic to plasma. 
U/P osmolality ratios approximating 1 have uni- 
formly been observed under circumstances in 
which the final urine is either hypotonic or hyper- 
tonic. These findings are consistent with results 
in other species and have been interpreted as indi- 
cating that the luminal membrane in the proximal 
nephron is freely permeable to water. Net reab- 
sorption of fluid, as evidenced by a progressive in- 



NATIONAL HEART INSTITUTE 



81 



crease in the concentration of inulin in the luminal 
fluid, is considered to be effected by active sodium 
reabsorption and osmotic flow of water along the 
gradient established by the prior solute removal. 
It had been assumed that this process is uninflu- 
enced by antidiuretic hormone and that approxi- 
mately the same fraction of glomerular filtrate is 
reabsorbed proximally both in water diuresis and 
in antidiuresis. However, the results of the most 
recent studies in the dog are inconsistent with this 
thesis. The U/P inulin ratio has been noted to 
be greater in antidiuresis than in water diuresis, 
indicative of enhanced reabsorption of proximal 
fluid in antidiuresis when ADH is maximal. 
Since, as indicated above, the U/P osmolality is 
the same in both situations, it is probable that the 
increase in the U/P inulin in antidiuresis is pro- 
vided by an acceleration in the transport of sodium 
and chloride out of the nephron into the surround- 
ing cortical tissue. Vasopressin is known to stimu- 
late sodium transport in other epithelial tissues 
(frog and toad skin and bladder) . Consequently, 
it has been concluded tentatively that an analogous 
effect occurs in the proximal nephron and accounts 
for the increased reabsorption noted in antidiure- 
sis in the dog. This is an important observation 
which requires further study. 

In association with the above studies bicarbo- 
nate reabsorption has also been examined. In 
the dog the concentration of bicarbonate in the 
most distal portion of the accessible part of the 
proximal segment is slightly less than that of 
plasma. Apparently, reabsorption of bicarbonate 
exceeds water reabsorption slightly if at all. This 
contrasts with observations in the rat in which 
the concentration of bicarbonate falls progres- 
sively throughout the nephron. These studies 
are also being pursued as is an examination of 
potassium reabsorption under a variety of experi- 
mental conditions in the intact dog. 

b. The Influence of Nonelectrolytes on Urine 
Concentration 

Urea is known to play a unique role in the mech- 
anism of urine concentration. Its administration 
results in the attainment of a higher urine os- 
molality than with other solutes. This enhance- 
ment of urinary concentrating ability is thought 
to depend upon the permeability of the luminal 
membrane of the collecting duct to urea and its 
accumulation in medullary tissue. In order to 



obtain further support for this thesis, the effect 
of other relatively diffusible nonelectrolytes on the 
concentrating mechanism was examined in the 
dog and correlated with the degree of accumula- 
tion of the nonelectrolyte in the medullary region 
of the kidney. Earlier work had established that 
certain urea analogues do, in fact, increase the 
ability of the rat to elaborate a hypertonic urine, 
though to a lesser degree than does urea. Similar 
studies in the dog have now been concluded, and 
it has been established that not only urea, but also 
methylurea, acetamide and 1,2- propanediol pro- 
duce enhancement of concentrating ability in this 
species. As with urea, all of the latter compounds 
diffuse across the luminal epithelium and accumu- 
late in highest concentration in the medullopapil- 
lary region of the kidney. Propanediol was the 
least effective compound studied with respect to 
enhancement, and in accordance with theory, ac- 
cumulates in the medulla to the smallest extent. 
On the other hand, its clearance relative to inulin 
is the lowest of the compounds studied, indicating 
that it must be reabsorbed to a considerable de- 
gree high in the nephron, presumably in the prox- 
imal segment. 

c. Measurement of Medullary Blood Floiv 

For some time efforts have been directed at 
developing a method for the measurement of 
medullary flow. The purpose of these studies was 
to examine the effect of changes in medullary 
blood flow on urine concentration in an effort to 
study the influence of the countercurrent system 
on the process. A number of methods have now 
been tested and discarded. Unfortunately, the 
characteristics of the countercurrent system of 
itself preclude use of the presently available meth- 
ods, all of which are based on the Fick principle 
which requires constancy of arterial concentration 
of the test subject. However, hi the course of 
these studies, a precise method for the measure- 
ment of regional tissue flow has been developed. 
The method utilizes the Fick principle and re- 
quires administration of hydrogen gas and esti- 
mation of the curve of desaturation of tissue hy- 
drogen gas with time. From the slope of this 
curve blood flow may be calculated. Hydrogen 
gas is measured by use of a platinum electrode. 
Its concentration in tissue is equal to that in the 
vein, thereby eliminating the necessity of simul- 
taneous venous determinations. Furthermore, the 



82 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



arterial concentration approaches zero within 
seconds after cessation of either intra-aortic ad- 
ministration of hydrogen or administration by 
respiration. Consequently, simultaneous arterial 
concentrations are also unnecessary. The sim- 
plicity of the method due to elimination of the 
necessity of simultaneous arteriovenous differences 
is remarkable. Thus far, the method has been 
tested successfully in cardiac and skeletal muscle, 
brain, renal cortex, etc. The results to date are in 
excellent agreement with those obtained by other 
conventional methods. 

II. Electrolyte and Water Transport Across Bio- 
logical and Artificial Membranes 

a. Electrolyte Fluxes in Separated Renal Tubules 
of the Rabbit 

Fluxes of sodium and potassium have been esti- 
mated in the past in thin slices of renal cortical 
tissue. Data from such studies have been difficult 
to interpret, since the anatomic arrangement of 
the cells in tissue slices complicates precise analy- 
sis. The fluxes obtained in this manner are merely 
gross estimates of the exchange of isotope into and 
out of the whole slice and provide no direct in- 
formation concerning the actual fluxes across the 
individual cell membranes. Although this diffi- 
culty has been recognized by most workers in the 
field, it had not been generally appreciated that 
the fluxes across whole slices may vary not only 
in a disproportionate manner from those across the 
cell membranes, but may also vary in direction 
during experimental manipulation. Last year a 
method was designed which eliminated some of 
these difficulties, and permitted relatively direct 
estimates of fluxes of electrolytes across individual 
cell membranes. The cortical tissue of rabbits 
was treated with collagenase, the resulting sepa- 
rated segments of proximal tubules were suspended 
in appropriate media, and flux measurements per- 
formed using sodium 24 and potassium 42 . Using 
this technique, a number of interesting and im- 
portant observations have been made within the 
past year. 

In the first place, the cell suspension as noted 
earlier, was shown to be viable as indicated by 
adequacy of respiration, maintenance of steady 
state concentrations of sodium, potassium, and 
tissue water, and the maintenance of the ability 
to transport and accumulate PAH actively. It has 



also been shown that potassium within the tubule 
cells may be divided kinetically into a minimum 
of two distinct compartments. Addition of the 
cardiotonic steriod strophanthidin, a known in- 
hibitor of active transport in other tissues as well 
as in the kidney, reduces the potassium content of 
the cell suspension. These results are similar to 
those observed in slices. The reduction in potas- 
sium content has been shown to be due, in part at 
least, to a specific decrease in the influx of potas- 
sium into the cell. In contrast to the results of 
slice studies, in which the efflux rate constant was 
either unaffected or actually increased following 
addition of strophanthidin, it has been demon- 
strated unequivocally that the efflux rate constant 
actually falls following addition of the drug. This 
difference in the results of the studies using tissue 
slices and cell suspensions emphasizes both the 
difficulties involved in the interpretation of flux 
studies in whole slices and the unique potentiality 
of the tubule suspension system for the analysis 
of fluxes across individual cell membranes. Most 
recently, it has also been established that at 0° C, 
a temperature at which it is generally assumed 
active transport ceases, all of the tissue potassium 
within the cell suspension remains exchangable. 
This, too, is at variance with earlier studies in 
slices. Of greater importance and also in contrast 
to the results of slice studies, it has been possible 
to demonstrate the persistence of active transport 
at this temperature. 

Studies Concerning the Exchange of Na 24 . 
Earlier slice studies had not revealed the pres- 
ence of a tissue sodium compartment responsive to 
known inhibitors of cation transport. This obser- 
vation, together with other negative findings, had 
led to the conclusion that sodium and potassium 
transport may not be linked in renal tubular cells. 
Using the present technique, it has been possible 
to analyze the tissue sodium compartments and ex- 
change rates with greater precision. At least two 
compartments are present in the tubule cells, one 
extremely rapid, equilibrium being virtually com- 
plete within 60 seconds, and the other a slow and 
more readily discernible compartment. The fast 
compartment, which exchanges at a rate of ap- 
proximately five times per minute, is responsive to 
strophanthidin and other inhibitors such as anoxia. 
The former compound increases the sodium con- 
tent of the tissue by reducing the rate constant 



NATIONAL HEART INSTITUTE 



83 



for sodium efflux. In view of this demonstration, 
it is probable that this rapid compartment, not 
clearly discernible in kinetic studies in slices, is of 
physiological significance. However, a 1 :1 link be- 
tween sodium and potassium transport, which had 
been assumed, was not demonstrable. Instead, 
sodium efflux appeared to be five to seven times 
greater than simultaneously estimated potassium 
influx. Despite this lack of stoichiometry, sodium 
efflux was shown to be dependent upon the pres- 
ence of potassium in the bathing solution. Efflux 
was significantly reduced when the medium po- 
tassium was decreased from 5 to .3 mM/L. 

Para-aminohippuric Acid Studies. PAH 
transport has also been examined in the tubule cell 
suspension system. These studies are incomplete 
but, as with the others reported above, the results 
differ significantly from those obtained in slices. 
Thus, for example, diodrast, a competitive inhibi- 
tor of PAH accumulation, had been assumed to 
lower the concentration of PAH in renal tissue by 
increasing the "run-off" or efflux of PAH from 
the cells. In view of the difficulties in interpreta- 
tion of efflux measurements in slices with respect 
to K alluded to above, it was not surprising to note 
in the present studies that diodrast clearly reduces 
the active uptake of PAH and does not influence 
the efflux rate constant. 

b. Studies in Red Cell Ghosts 

The red cell ghost system has been described in 
detail in earlier reports. Its advantage as a model 
for the study of electrolyte transport has been dis- 
cussed. The most recent experiments were de- 
signed to determine whether the red cell 
membrane, prepared by fragmenting hemolyzed 
red cells, selectively binds electrolytes and non- 
electrolytes. The fragmented membrane was dia- 
lyzed against various solutions at 37°. Radio- 
active tracers of sodium, potassium, chloride or 
glucose were added to the outside bathing medium 
and binding estimated from the ratio of the counts 
within the dialysis bag to those outside. Electro- 
lyte "binding" was studied in rat red cell mem- 
branes, glucose "binding" in human red cell mem- 
branes. Although in the last report it had been 
suggested that the rat red cell may selectively 
"bind" potassium on the outer surface, no selective 
binding was observed in the present studies. In- 
stead a Donnan distribution of sodium and potas- 
sium was noted. Of interest, however, was the fact 

705-685—63 7 



that the Donnan ratios were reduced by the addi- 
tion of the divalent cations, magnesium or calcium. 
The magnesium and calcium effect is inhibited by 
the addition of EDTA, a potent chelating agent, to 
the medium, as well as by the addition of ATP. 
The effect of the ATP is presumably due to its 
ability to act as a chelating agent and is not spe- 
cifically related to the provision of high energy 
phosphate, as has been suggested by others. 

The experiments involving glucose binding in 
human red cells are in a preliminary form at the 
present time. Since the isotope method selected 
for the demonstration of binding assumes no meta- 
bolic transformation of glucose, final interpreta- 
tion of the observations will require additional 
study. Recognizing this importnat qualification, a 
number of observations of interest have been made. 
Glucose (or at least label) is bound or accumulates 
within the dialysis bag in association with the 
fragmented membrane. The "binding" requires 
the presence of ATP and magnesium. Unlabeled 
glucose or sorbose, but not fructose, arabinose, 2- 
deoxyglucose or mannose prevent or decrease the 
apparent glucose "binding." It is of interest of 
that C 1 * sorbose also "binds" in the presence of 
ATP and magnesium. No interpretation of these 
studies is as yet available, since it depends, as in- 
dicated above, on the final determination of the 
metabolic state of the administered C 14 glucose. 

Measurement of Cytoplasmic and Nuclear 
Membrane Properties or Amphiuma Erythro- 
cytes. The purpose of these studies was to meas- 
ure the potential difference across both the cyto- 
plasmic and nuclear membranes of Amphiuma red 
cells. Amphiuma red cells were selected because 
of their large size and consequent suitability for 
micropuncture analysis. The cells contain ap- 
proximately 70% water, 11.0 mM of sodium, and 
87 mM of potassium per liter. As a preliminary 
to the electrical measurements, fluxes of potassium 
and sodium have been estimated. Sodium efflux is 
inhibited by cardiac glycosides, by dinitrophenol 
and by removal of potassium from the medium. 
The inside-outside concentration ratio for chlo- 
ride has been established, and it has been assumed 
that the potential across the cytoplasmic mem- 
brane would be a reflection of the chloride diffu- 
sion potential. However, to date it has not been 
possible to obtain data in support of this assump- 
tion. "Whether the potentials measured are ac- 



84 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



curate reflections of the true potential or represent 
methodological difficulties has not yet been estab- 
lished. 

III. Mechanism of Action of Vasopressin and 
Other Hormones 

a. Vasopressin 

Antidiuretic hormone increases the permeability 
of a number of biological membranes to water. Its 
role in the elaboration of a hypertonic urine of 
low volume is well known, and depends upon hor- 
mone-induced enlargement of aqueous channels 
or pores in the tubular epithelium, which permits 
osmotic flow of water out of the distal nephron 
into the surrounding interstitial tissue. On the 
basis of studies presented last year, it has been 
proposed that the hormone exerts its effect in re- 
sponsive tissues (toad blader, kidney) by stimu- 
lating the production and/or accumulation of cy- 
clic AMP in the tissue. The nucleotide is thought 
to alter membrane permeability either directly, or 
more likely, by initiating a series of unknown re- 
actions in the tissue. Much of the work in the 
past year has been directed at developing addi- 
tional evidence in favor of the thesis. 

In collaboration with a group at Western Re- 
serve University, the activity of cyclic AMP in 
toad bladder tissue has been measured with and 
without hormone. Thus far it has been shown 
that the nucleotide is present in the epithelial 
tissue of the toad bladder. Although antiduretic 
hormone increased the concentration of cyclic 
AMP in some studies, it has not yet been possible 
to demonstrate a reproducible rise in the concen- 
tration of the nucleotide in the toad bladder tissue 
following incubation with hormone. On the other 
hand, theophylline, which prevents the degrada- 
tion of cyclic AMP to its inactive form, 5' AMP, 
uniformly increases the concentration of the cyclic 
form in the tissue. The methylxanthines had been 
shown to exert in toad bladder effects on water 
and sodium transport which are indistinguishable 
from those produced by antidiuretic hormone. 

In association with the above studies the meta- 
bolic effects of vasopressin and of the proposed 
intermediate in its action, cyclic AMP, are being 
examined in both toad bladder and renal tissue. 
Others have shown that the hormone increases 
oxygen consumption and glycogenolysis in toad 
bladder when incubated in sodium-containing 



medium. These results have been confirmed. In 
addition, cyclic AMP increases oxygen consump- 
tion and glycogenolysis, whereas the degradation 
product, 5'AMP, has no effect or depresses gly- 
cogenolysis. Similar studies are now being car- 
ried out using dog renal cortical tissue. 

Phosphofructokinase activity has also been 
assayed in the toad bladder. Cyclic AMP is 
known to stimulate the activity of this enzyme in 
tissues in which it is the rate-limiting enzyme in 
glycogenolysis. The enzyme apparently is not 
rate-limiting in the toad bladder, and although 
some stimulation is produced by both cyclic AMP 
and its degradation product, 5'AMP, vasopressin 
is without effect. 

The most interesting evidence concerning the 
relationship between cyclic AMP and antidiuretic 
hormone concerns their respective effects on phos- 
phorylase activity in toad bladder and renal 
tissue. In virtually all tissues examined, cyclic 
AMP increases the activity of glycogen phos- 
phorylase. It has now been shown that a similar 
effect occurs in toad bladder, and of greater sig- 
nificance, it has been possible to demonstrate a 
uniform increase in glycogen phosphorylase activ- 
ity in toad bladder following incubation with 
vasopressin and other neurohypophyseal hor- 
mones. Epinephrine and ACTH, which increase 
phosphorylase activity in muscle and adrenal re- 
spectively, are without effect in toad bladder. 
Further evidence concerning the relationship be- 
tween the hormone and its supposed intermediate 
has been developed by showing that acidification 
of the bathing medium, a procedure which inhib- 
its the permeability effect of ADH also prevents 
the hormone-induced increase in phosphorylase 
activity. Most recently it has been possible to 
show that phosphorylase activity in both rabbit 
renal medullary tissue and dog cortical and med- 
ullary tissue are significantly increased by ADH. 
This represents the first demonstration of a 
specific metabolic effect of the hormone in mam- 
malian renal tissue. Although these results lend 
support to the view concerning the intermediacy 
of cyclic AMP in ADH action, they do not afford 
any insight into the precise mechanism of action 
of the hormone on membrane permeability. 

Other investigators have suggested that the 
action of vasopressin is dependent upon prior link- 
age of the octapeptide via its disulfide bridge to 
sulfhydryl groups on the membrane. Support for 



NATIONAL HEART INSTITUTE 



85 



this hypothesis derives from studies in which cer- 
tain reducing agents (cysteine and thioglycollate) 
have been shown to prevent the permeability effect 
of the hormone. These results are difficult to inter- 
pret, however, since the octapepticle remains in the 
reduced form only in the presence of the reducing 
agent. Consequently, it is not possible to distin- 
guish between an effect of the reducing agent on the 
tissue or on the octapeptide or on both. The inter- 
pretation is further complicated by recent results 
obtained in this laboratory. Both cysteine and 
thioglycollate interfere with the permeability 
effect of vasopressin and theophylline, but do not 
alter the response to cyclic AMP. Clearly the 
effect of theophylline cannot involve the postulated 
disulfide bond, nor can significant alterations of 
the sulfhydryl groups on the membrane account 
for the persistence of the cyclic AMP effect. It 
has been suggested tentatively that these reducing 
agents may actually interfere with the production 
of cyclic AMP in the tissues, Both antidiuretic 
hormone, which is thought to stimulate its produc- 
tion, and theophylline, which prevents degrada- 
tion of the intermediate, would be expected to be 
unaffected in the presence of an agent which limits 
the enzymatic conversion of ATP to cyclic AMP, 
whereas the effect of exogenous cyclic AMP should 
be unaltered by the reducing agent. 

h. Aldosterone and the Renin-Angiotensin System 

An examination of the factors involved in the 
control of aldosterone secretion by the adrenal con- 
tinues. Last year it was unequivocally demon- 
strated that the kidney secretes a substance, now 
known to be renin, which stimulates aldosterone 
release from the adrenal. During the past year 
further suppoxi: for this observation has been ob- 
tained. Angiotensin-like activity in lymph from 
normal dogs has been assayed and compared with 
similar analyses of lymph from dogs with experi- 
mental hyperaldosteronism due to caval constric- 
tion. Angiotensin-II activity, a reflection of the 
amount of renin released from the kidney, was con- 
siderably greater in lymph from caval animals 
than from normals. A double assay system was 
used in these studies, the first, the pressor effect 
of the extract in appropriately treated rats, and 
the second, the steroidogenic response of the iso- 
lated adrenal of hypophysectomized-nephrec- 
tomized dogs. The results were similar using both 



methods of assay, in that lymph of the caval ani- 
mals increased blood pressure in rats and increased 
aldosterone secretion of the isolated adrenals to a 
greater extent than did lymph from normal 
animals. 

In association with members of the Cancer In- 
stitute, it has been proven that Sprague-Dawley 
rats bearing Walker's carcinosarcoma-256 secrete 
aldosterone at an excessively high rate. These rats 
were noted in the past to retain sodium, leading 
to the suspicion, now confirmed, that aldosterone 
secretion is elevated. On the other hand, corti- 
costerone secretion was unaltered in these animals. 

Alterations in plasma sodium and potassium 
concentrations are also known to affect aldosterone 
secretion in a number of species. Observations 
similar to those noted in other laboratories have 
been extended in an effort to characterize the 
mechanism more clearly. Thus, it has been ob- 
served that the intravenous injection of potassium 
chloride or of K 2 S0 4 into hypophysectomized dogs 
results in a striking increase in aldosterone secre- 
tion. Corticosterone secretion also rose in these 
studies. In three of six animals, despite nephrec- 
tomy, hypersecretion of aldosterone continued 
during maintenance of hyperkalemia, indicating 
that in these animals neither the renin-angiotensin 
system nor the presence of an intact anterior 
pituitary was essential for aldosterone release. 
Furthermore, in agreement with other studies, the 
direct injection of potassium chloride or potassium 
sulfate into the isolated adrenal also increased 
aldosterone release, supporting the view that po- 
tassium exerts a direct effect on steroidogenesis 
within the adrenals. Preliminary studies have 
been interpreted as indicating that a reduction in 
the plasma sodium concentration effected by dilu- 
tion may also stimulate release of aldosterone 
from the adrenal. 

The effect of prolonged and continuous intra- 
venous infusion of angiotensin-II in unrestrained 
normal dogs has been studied. The most impor- 
tant observation thus far noted is that rates of 
angiotensin-II infusion, insufficient to elevate 
blood pressure in the unrestrained animal, result 
in enhanced urinary aldosterone excretion. How- 
ever, angiotensin also produced transient renal 
hemodynamic effects which apparently exerted 
a direct influence on electrolyte excretion. These 
studies are being continued. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



IV. Cardioglobulin 

As discussed in detail in previous reports, 
plasma is known to contain a protein system com- 
posed of at least three globulin fractions, which 
exerts an inotropic effect on the isolated frog 
heart. This system is present in mammalian 
plasma and may play an important physiological 
role in the maintenance of normal cardiac con- 
tractility. In the past year the chemistry of this 
system has been intensively studied. It has been 
observed that one of the components of this sys- 
tem, cardioglobulin A, is rapidly inactivated by 
a variety of tissue homogenates as well as by 
plasma. The inactivation has the characteristics 
of an enzymatic process, and has been shown to 
involve the release of inorganic phosphate. Inac- 
tivation is prevented by the addition of certain 
high energy phosphate compounds, including 
ATP, ADP and creatinine phosphate. On the 
basis of these observations it has been tentatively 
concluded that cardioglobulin A contains a high 
energy phosphate which is removed by a plasma 
and/or a tissue enzyme, thereby accounting for 
its inactivation. Furthermore, on the basis of 
other information, it has been suggested that the 
high energy phosphate in cardioglobulin A may 
provide energy, not for the contractility effect di- 
rectly, but for the transport of a cardioglobulin C 
calcium complex into heart muscle. Further sup- 
port for the view that a high energy phosphate is 
associated with cardioglobulin A was the observa- 
tion that the gradual and progressive decline of 
activity of the entire cardioglobulin system in the 
assay system in frog heart is associated with re- 
lease of inorganic phosphate into the medium and 
a disappearance of cardioglobulin A activity. 

In association with the above studies, an attempt 
at obtaining stable fractions of the three cardio- 
globulin components is under way. It has been 
observed that a cardioglobulin inhibitor is elab- 
orated in plasma during the usual preparative 
separation of the three compounds. The forma- 
tion of the inhibitor has unfortunately further 
complicated the fractionation system, and studies 
at the present time are directed at eliminating this 
particular problem. It is of interest that the for- 
mation of the so-called inhibitor in rat plasma is 
prevented by the addition of sodium ascorbate, 
but not by the addition of other reducing agents. 
Vitamin C, on the other hand, does not prevent 



the development of the so-called inhibitor during 
the fractionation process itself. 

LABORATORY OF METABOLISM 

Before presenting details it may be useful to 
call attention to some selected areas in which par- 
ticularly significant progress has been made dur- 
ing the past year. 

An important advance has been the definitive 
demonstration of a hormone-stimulated lipase in 
adipose tissue. The magnitude of the effects ob- 
tained adequately accounts for the observed in- 
creases in rate of release of fatty acids during hor- 
monal stimulation. At the same time, using a 
newly developed balance method, it has been 
shown that fatty acid esterification is actually 
increased during hormonal stimulation of fatty 
acid release. Consequently, it can now be con- 
cluded that the primary mechanism for increasing 
rates of fat mobilization lies in a system capable 
of rapidly activating one of the lipases of adipose 
tissue (sec. 2). 

On the basis of previous studies we have sug- 
gested that basal metabolic rate might be signifi- 
cantly influenced by rates of free fatty acid 
(FFA) mobilization and we put forth the tenta- 
tive hypothesis that the hypermetabolism of the 
hyperthyroid state might be secondary to an ab- 
normally high rate of fatty acid mobilization. 
Important new evidence has now been obtained 
from clinical studies that supports this hypothesis. 
It has been shown that the administration of hexa- 
methonium, which inhibits FFA release from adi- 
pose tissue, has a small but definite depressing 
effect on the metabolic rate of normal subjects that 
have been made hyperthyroid by treatment with 
triiodothyronine. It has also been shown that 
intravenously administered norepinephrine raises 
the metabolic rate of normal subjects while simul- 
taneously increasing the turnover of serum FFA. 
Pretreatment with an adrenergic blocking agent 
abolished both the stimulation of FFA turnover 
and the hypermetabolic effect of the norepi- 
nephrine (Section #4). 

Further studies on cholesterol biosynthesis have 
shown that reduction of the side-chain double 
bond probably occurs at several points. Dihy- 
drolanosterol and desmosterol appear to be rele- 
gated to relatively minor roles in the normal path- 
way. From studies with triparanol and other 
similar inhibitors and from studies of the proper- 



NATIONAL HEART INSTITUTE 



87 



ties of "desmosterol reductase" it has been con- 
cluded that a single enzyme in liver particles is 
actually responsible for reduction of the side-chain 
independent of the structure of the sterol nucleus. 
In other words, we now believe that liver contains 
a "side-chain reductase" with only limited specific- 
ity. Triparanol inhibits this reductase and thus 
blocks reduction of the side-chain at all stages, 
leading to the previously reported accumulation 
of desmosterol (sec. 1-B). 

1. Pathway of Cholesterol Biosynthesis and In- 
hibitors of Cholesterol Biosynthesis 

(A ) Normal Pathway of Cholesterol Biosynthesis 

Development of a powerful technique for frac- 
tionation of sterols by thin layer chromatography 
has made possible a series of studies of sterol me- 
tabolism in the liver and in the skin. This thin 
layer technique permits rapid and quantitative 
resolution of a number of sterol intermediates 
that previously could be isolated only by laborious 
and time-consuming column techniques or by gas- 
liquid chromatographic analysis of only very 
small samples. 

Studies of the time course of incorporation of 
radioactive mevalonic acid into the liver sterols 
of intact rats have yielded valuable evidence re- 
garding the normal pathway of cholesterol metab- 
olism. One school of investigators has maintained 
that the side-chain double bond of lanosterol is 
retained until the very last step in cholesterol 
biosynthesis. Others, on the basis of isotopic data, 
have shown that sterols with saturated side-chains 
can be converted to cholesterol and suggested that 
reduction of the side-chain of lanosterol might 
occur as the very first step after cyclization of 
squalene. The present studies show that very 
little radioactivity is observed in dihydrolanos- 
terol and likewise very little is observed in desmos- 
terol during the first 30 minutes after injection 
of labeled mevalonate. Since major amounts of 
radioactivity were found in A 7 ( + A 8 ) -cholestenol, 
it is concluded that side-chain reduction occurs 
prior to the shift of the nuclear double bond to the 
A 5 position. A fraction of C 28 sterols was isolated 
and found by chromatographic analysis to contain 
a mixture of molecules with saturated and un- 
saturated side-chains. Since this fraction con- 
tained considerable radioactivity, it is probable 
that side-chain reduction occurs to some extent at 



the C 28 stage. In summary, the evidence obtained 
supports the suggestion previously made that there 
is not a single unique pathway of cholesterol bio- 
synthesis in normal rat liver. Instead it appears 
that reduction of the side-chain double bond can 
and does occur at several different stages during 
modification of the sterol nucleus. The present 
findings indicate that reduction at the very first 
step (lanosterol to dihydrolanosterol) and at the 
very last step (desmosterol to cholesterol) are 
relatively minor pathways. 

(B) Desmosterol Reductase 

Studies completed this year support the con- 
clusion, made previously, that the enzyme respon- 
sible for reduction of the side-chain double bond 
in desmosterol also catalyzes the reduction of the 
side-chain double bond in lanosterol, A 7,24 -choles- 
tadienol and probably in other intermediates. In 
other words, it appears that the enzyme, while 
apparently rather specific for the sterol side-chain 
is relatively nonspecific as regards the nuclear 
configuration of the sterol. 

The evidence leading to this conclusion is briefly 
as follows : reduction of lanosterol to dihydro- 
lanosterol in a cell free system was demonstrated 
for the first time. It was shown that "lanosterol 
reductase" activity was located in the particulate 
fraction of liver, both in microsomes and in mito- 
chondria. The enzyme is TPNH specific and it 
is inhibited by reagents that interact with sulfhy- 
dryl groups. In all of these respects "lanosterol 
reductase" activity is essentially identical to 
"desmosterol reductase" activity. Furthermore, 
triparanol and two other inhibitors of desmosterol 
reduction were shown to inhibit lanosterol reduc- 
tion when added to homogenates. 

Evidence was obtained from in vivo studies that 
triparanol inhibits reduction of A 7,24 -cholestadienol 
and zymosterol. When labeled mevalonate was 
administered to triparanol-treated rats significant 
amounts of radioactivity were found in these 
sterols with unsaturated side-chains but in normal 
rats the radioactivity was mostly associated with 
the corresponding saturated side-chain sterols. 

We are then led to conclude that what we have 
previously called "desmosterol reductase" should 
now be regarded as a generalized "side-chain re- 
ductase." Triparanol and several other inhibitors 
block the activity of this reductase and lead to the 
accumulation of desmosterol. As was pointed out 



88 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



in earlier publications, the accumulation of des- 
mosterol under these circumstances does not prove 
that it is necessarily a major intermediate in the 
untreated animal. The work described above does 
in fact appear to relegate desmosterol to a minor 
role while at the same time confirming that it is 
one of the precursors of cholesterol. 

(C) Desmosterol as a Precursor of Adrenal 
Steroids and Bile Acids 

As reported last year, clinical studies provided 
evidence that desmosterol could be directly con- 
verted by man both to bile acids and to adrenal 
steroids. Further evidence for this direct conver- 
sion has now been obtained in mice and rats by in 
vitro techniques. It has been shown that homog- 
enates of adrenal gland can convert desmosterol 
to corticosteroid (compound B) and that this con- 
version does not go through cholesterol. Using 
liver homogenates it has been shown that the side- 
chain of desmosterol can be readily oxidized. The 
rates of metabolism of cholesterol and of desmos- 
terol were compared in both the adrenal homoge- 
nates and in preparations of liver mitochondria. 
The two sterols were metabolized at comparable 
rates. Whether or not desmosterol is an interme- 
diate in the conversion of cholesterol to steroids 
and bile acids is not known but these findings are 
compatible with such a possibility. 

(D) Sterol Metabolism in Skin and in the Optic 
Lens 

A comprehensive survey of skin sterols in nor- 
mal animals and in triparanol treated animals was 
carried out. It appears that the mechanism of 
action of triparanol in the skin is similar to that in 
the liver, that is, it exerts a general inhibition of 
side-chain reduction. Simultaneous studies of 
pathological changes in the skin were carried out 
in collaboration with Dr. Wertlake in the Patho- 
logical Anatomy Department, Whether the de- 
generation and atrophy observed is attributable 
to the desmosterol accumulation demonstrated can- 
not be stated but further studies along these lines 
may be fruitful. 

It was shown that desmosterol accumulates in 
the optic lens of triparanol-treated rats. The 
structural changes in the lens have been studied by 
optical and electron microscopy by Dr. von Sall- 
mann in the Ophthalmology Branch of NINDB. 



{E) Atherogenicity of Desmosterol 

It was shown for the first time that desmosterol 
is deposited in the atherosclerotic lesions and de- 
posited to about the same extent as cholesterol. 
Because of the limited availability of desmosterol 
only one experiment was possible but the results 
of this critical study were clear cut. A rabbit was 
put on anatherogenic diet containing cholesterol 
and also desmosterol. After about four months on 
the diet the animal was found to have gross ather- 
oscelrotic lesions. At the time of sacrifice desmos- 
terol accounted for about one-third of the circu- 
lating sterols. Again, one-third of the sterols in 
the atherosclerotic lesions was found to be des- 
mosterol and two-thirds cholesterol. Thus it has 
been shown that if desmosterol is present during 
the genesis of an atherosclerotic lesion it will be 
deposited at no greater and no less a rate than 
cholesterol. Postmortem studies reported from 
other laboratories have been interpreted to show 
that desmosterol might be less atherogenic than 
cholesterol because only a small amount of desmos- 
terol was found in lesions of patients that had been 
treated with triparanol for a few months before 
death. Because the lesions in these cases were un- 
doubtedly well advanced at the time triparanol 
treatment was instituted it would be anticipated 
that desmosterol would constitute only a small 
fraction of the total sterol in the lesions, inasmuch 
as the increment in sterols laid down during the 
relatively short period of triparanol treatment 
must have been quite small. The present animal 
study must give a more relevant result since des- 
mosterol was present in the serum throughout the 
period during which the lesions developed. 

2. The Metabolism of Adipose Tissue as In- 
fluenced by Hormones 

The mobilization of fatty acids from adipose 
tissue is a key process in the control of energy 
metabolism in the fasting state and in response 
to exercise. An understanding of the hormonal 
factors controlling this process will undoubtedly 
be as important as the understanding of factors 
controlling glucose mobilization and utilization. 

An important advance has been made in this 
area during the past year. It has been shown that 
under the proper conditions a lipase system in 
adipose tissue can be dramatically activated when 



NATIONAL HEART INSTITUTE 



89 



the tissue is exposed even for only a few minutes 
to epinephrine, norepinephrine, glucagon or 
ACTH. Previous reports have appeared from two 
laboratories showing that 25 to 50% increases in 
lipase activity could be effected with these hor- 
mones but the rate of fat breakdown in the intact 
tissue increases by several fold. The quantitative 
disparity left doubt as to the significance of this 
hormone-stimulated lipase activation. We have 
now been able to show increases in lipase activity 
of a magnitude adequate to explain the increased 
rates of fat mobilization. 

A finding of great value in the further develop- 
ment of this problem is that the major fraction 
of lipase activity in adipose tissue homogenates 
is intimately associated with fat and can be sep- 
arated from the bulk of the cell material by float- 
ing it to the top of a centrifuge tube. This should 
make the further purification and characterization 
of the enzyme a much easier problem. 

A method has been developed for simultaneous- 
ly determining rates of lipolysis and rates of es- 
terification of fatty acids in intact adipose tissue. 
This method appears to be free of the ambiguities 
associated with isotopic techniques for measure- 
ment of the rate of these processes. By applying 
this method to tissue stimulated by the lipolytic 
hormones it has been shown that parallel with the 
increase in lipolytic activity there is a marked 
increase also in the rate of fatty acid esterifica- 
tion. The latter may be an indirect effect second- 
ary to the increase in intracellular fatty acid con- 
centration but this is not yet firmly established. 

It has been demonstrated for the first time that 
adipose tissue contains, in addition to the 
hormone-sensitive lipase discussed above, lipase 
with very high activity in the splitting of mono- 
gycerides. The latter enzyme is not affected by the 
hormones that increase fat release from adipose 
tissue. 

1 Prostaglandin is an acidic lipid first identified 
in extracts of seminal fluid and prostate gland 
which has potent vasodepressor activity. Last 
year it was obtained in crystalline form and its 
structure determined by Dr. Sune Bergstrom and 
coworkers at the Karolinska Institute in Stock- 
holm (2-(6-carboxyhexyl)-3- (3-hydroxyocten-l- 
yl)-4-hydroxycyclopentanone). Several prosta- 
glandin derivatives were obtained from Dr. 
Bergstrom and examined for possible effects on 
adipose tissue metabolism. It was shown that 



prostaglandin E has a slight inhibitory effect on 
lipolysis in adipose tissue. More striking was the 
ability of prostaglandin to counteract the stimula- 
tory effect of epinephrine and other lipolytic hor- 
mones on fat breakdown in the epidymal fat pad. 
It was further shown that in intact dogs prosta- 
glandin counteracts the vasopressor effects of 
epinephrine and norepinephrine. These findings 
suggest the possibility that the biochemical basis 
for the vasodepressor effects of prostaglandin and 
its ability to partially block the action of lipolytic 
hormones may be related. Further studies are in 
progress along these lines. The physiological role 
of prostaglandin remains to be established but the 
fact that it has now been shown to occur in other 
tissues as well as in the accessory sexual glands 
makes it possible to visualize a hormonal function 
for this material. 



3. Studies on the Factors Controlling Mobiliza- 
tion and Utilization of Free Fatty Acids 
(FFA) in Vitro 

Previous work from this laboratory showed that 
the rate of utilization of labeled fatty acids by 
muscle and by liver in vitro increases with the con- 
centration of fatty acids in the medium. These 
studies have now been extended by using a per- 
fused liver system. It was shown that the uptake 
of FFA was greatly increased when the concentra- 
tion of FFA in the perfusing fluid was high. An 
important additional observation was that the 
oxygen consumption by the perfused liver in- 
creased when it was perfused with high concen- 
trations of FFA. All of these studies suggest that 
utilization of this substrate is a function of the 
concentration at which it is offered to the periph- 
eral tissues. On the other hand, the in vitro 
studies, while they show an increased utilization 
of labeled fatty acids, fail to show any effect of 
FFA concentration of Q o2 . Attempts are being 
made to determine whether serum factors or hor- 
monal factors are necessary for optimal FFA 
utilization. 



4. Clinical Studies of the Relation Between FFA 
Utilization and Metabolic Rate 

A series of clinical studies has now been com- 
pleted with respect to the importance of FFA con- 
centration as a factor determining basal metabolic 



90 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



rate. These studies were done in collaboration 
with. Dr. Elsworth Buskirk and Dr. Ronald 
Thompson, making use of the metabolic chamber. 
It has been shown that intravenously administered 
norepinephrine consistently produces an increase 
in oxygen consumption — 10 to 20% above control 
values. This increase in oxygen consumption is 
associated with the expected rise in serum FFA 
concentrations and an increase in serum FFA turn- 
over, as determined from measurements of pal- 
mitate-1-C 14 turnover. When an adrenergic block- 
ing agent (pronethalol) was given just prior to the 
intravenous administration of norepinephrine, the 
rise in serum FFA levels was prevented and there 
was no increase in oxygen consumption. In fact, 
under the influence of the blocker, norepinephrine 
actually caused a slight but definite decrease in 
oxygen consumption. 

We have previously postulated that the hyper- 
metabolism seen in hyperthyroid patients might be 
secondary to a high rate of FFA mobilization. It 
has been previously shown that the adipose tissue 
of hyperthyroid animals releases fatty acids at a 
higher than normal rate and that adipose tissue 
from hyperthyroid animals shows a much greater 
response to lipolytic hormones. The question 
posed was whether interference with FFA mobili- 
zation in the hyperthyroid patient would influence 
his metabolic rate. Normal controls were made 
hyperthyroid by treatment with triiodothyronine 
(T 3 ). In the metabolic chamber the oxygen con- 
sumption was measured before and during the in- 
travenous administration of hexamethonium. Al- 
though the effects of hexamethonium were small, 
they were remarkably consistent, causing in every 
case a decrease in oxygen consumption of approxi- 
mately 10%. 

The results of these clinical studies coupled with 
the results of in vitro studies of FFA utilization 
are compatible with the hypothesis that mobiliza- 
tion of FFA may be one of the factors in determing 
basal metabolic rate. The general problem of how 
thyroid hormone increases body metabolism is now 
being reevaluated by this laboratory in the light of 
these findings. 

5. Differences in the Metabolic Fate of Fatty 
Acids of Different Structure 

It is now well established that dietary fatty 
acids of different structure influence serum cho- 



lesterol very differently. The biochemical basis 
for these effects of dietary fat has not yet been 
elucidated. Differences in the uptake of different 
labeled fatty acids are being explored. 

(A) Chylomicrons containing a predominantly 
saturated pattern of triglycerides are removed 
from the circulation more rapidly than chylomi- 
crons containing predominantly unsaturated tri- 
glycerides. This differential uptake was demon- 
strated in perfused livers as well as in the intact 
animal. On the other hand, there was no such 
differential uptake in perfused adipose tissue. 

Livers were perfused with high concentrations 
of linoleate or palmitate or both. These perfu- 
sions caused a net increase in the triglyceride and 
phospholipid content of the liver. The net changes 
were independent of which fatty acid was used 
but the distribution of fatty acids in the liver 
lipids depended upon the nature of the fatty acid 
used. Thus, the triglyceride fraction in the liver 
showed a doubling of the percentage content of 
palmitate when palmitate alone was perfused 
through the liver; when linoleate was used there 
was some increase in the linoleate content of the 
triglyceride fraction but to a smaller extent. Evi- 
dently the liver is able to accommodate increases 
in the amount of palmitate delivered to it more 
readily. This may be related to the specificity of 
the enzymes involved in triglyceride biosynthesis. 
In most animal species both the a and the a posi- 
tions tend to contain predominantly unsaturated 
fatty acids. 

C 14 -labeled fatty acids were used to compare the 
fates of linoleate and palmitate. Linoleate was 
preferentially incorporated into the phospholipid 
fraction, while palmitic acid was preferentially in- 
corporated into triglycerides and cholesterol esters. 

(B) The unsaturated fatty acids synthesized by 
normal animals are of the cis and trans isomers 
of oleic acid and of linoleic acid. It was found 
that absorption from the intestinal tract in the 
form of chylomicron triglycerides occurred at the 
same rate for each member of the isomeric pairs. 
Similarly, the rate of disappearance of the isomers 
from the circulation after injection of the free 
fatty acid occurred at essentially identical rates. 
Liver lipids were analyzed and again no difference 
was found for the rates of incorporation into cho- 
lesterol esters, triglycerides, diglycerides, mono- 
glycerides or phospholipids. In summary, then, 
oleic acid and its trans isomer (elaidic acid) and 



NATIONAL HEART INSTITUTE 



91 



linoleic acid and its trans, trans isomer appear to 
be indistinguishable, at least in terms of the rates 
at which they are metabolized in vivo. 

6. Clinical Studies of Serum Lipoproteins and 
Their Metabolism 

(A) The discovery of a syndrome characterized 
by almost complete absence of high density lipo- 
protein (HDL) from the serum affords an oppor- 
tunity to explore the function of this lipoprotein. 
The first patients were found living in an inbred 
population on the island of Tangier and the syn- 
drome has been named Tangier disease. Epidemi- 
ologic studies suggest that a single pair of allelic 
genes may exert major control over plasma HDL 
concentration. Patients with the rare homozygous 
defect have little or no plasma HDL, low serum 
cholesterol levels and infiltration of the RE system 
with cholesterol esters. The question of whether 
HDL plays a role in hypertriglyceridemia is being 
explored. 

(B) A standard procedure has been developed 
for determination of lipoprotein lipase levels in 
human serum. This procedure gives reproducible 
results and eliminates errors due to effects of en- 
dogenous substrate and nonlinearity that have 
been encountered with other methods. Applying 
this procedure to heparin-stimulated lipoprotein 
lipase release, it has been shown that most hyper- 
lipemic subjects have a normal response. The 10% 
with abnormal responses were patients having the 
"fat-induced" form of hyperlipemia. 

Serum glyceride levels determined by a direct 
method have been analyzed in over 300 fasting 
subjects to obtain satisfactory reference levels. 
The upper level of normal is established at 180 
mg/100 ml. 

A pedigree has been identified with hyperglyc- 
eridemia that falls neither in the fat-induced nor 
carbohydrate-induced category. The patients 
have normal cholesterol levels and normal lipopro- 
tein lipase activity. 

(C) There is good evidence that the triglycer- 
ides of lipoproteins or chylomicrons taken up by 
the liver are hydrolyzed and the free fatty acids 
subsequently incorporated into liver esters. Stud- 
ies of lipase activity in the liver have been ini- 
tiated. It has been shown that at least two dif- 
ferent lipases are present. The lipases split tri- 
glyceride emulsions in the absence of any serum 



activator. Partial purification of a lipase from 
the soluble fraction has been accomplished. 

7. The Metabolism of Cholesterol Esters 

Studies done in collaboration with Dr. Arthur 
Karmen of the Laboratory of Technical Develop- 
ment were undertaken to establish whether or not 
the formation of cholesterol esters during absorp- 
tion from the intestine showed selectivity. Mix- 
tures of labeled fatty acids were fed and the lipids 
recovered from chyle were extensively fraction- 
ated and studied. It was shown that the different 
fatty acids were incorporated in a nonselective 
way into triglycerides. On the other hand, oleic 
acid was used to a greater extent in the formation 
of cholesterol esters than was palmitic acid, lino- 
leic acid or stearic acid. Lecithin synthesized dur- 
ing absorption preferentially incorporated stearic 
acid, whereas oleic acid and palmitic acid were 
incorporated to a much smaller extent. 

The cholesterol esterase activity of rat liver has 
been studied with regard to cellular distribution 
and fatty acid specificity. Most of the enzyme 
was found in the soluble fraction with 11 to 30% 
in the microsomes. The hydrolysis of various 
cholesterol esters was studied and it was found 
that cholesteryl oleate and cholesteryl linoleate 
were hydrolyzed most rapidly, cholesteryl acetate 
occupied an intermediate position, while choles- 
teryl palmitate and cholesteryl stearate were least 
well hydrolyzed. The enzyme in the microsomal 
fraction appears to be different from that in the 
soluble fraction in certain properties, although the 
specificity of the two seems to be similar. 

The cholesterol ester synthesizing system of 
liver was studied. It was shown that both ATP 
and Coenzyme A are essential and that the syn- 
thesizing system is entirely particulate, being 
found both in the microsomal and mitochondrial 
fractions of liver. 

8. Studies on Experimental Nephrosis 

It is well known that the injection of anti-kid- 
ney serum into experimental animals can produce 
a syndrome similar to clinical nephrosis. The na- 
ture of this process has been studied using anti- 
body proteins that have been degraded by proteo- 
lytic enzymes by the methods described by Porter. 
The fragments obtained retain their ability to 
combine with kidney antigens but they do not in- 



92 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



duce proteinuria. It has been reported that the 
combination of different univalent fragments with 
antigen does not result in fixation of complement. 
The findings suggest that complement fixation is 
somehow essential for the processes that lead to 
damage of the glomerulus and to the full blown 
nephrotic syndrome. If this explanation is cor- 
rect, it should be possible to protect against kidney 
damage due to anti-kidney serum by lowering the 
levels of complement in the recipient animal. This 
possibility is being further explored. These stud- 
ies are being carried out in collaboration with Dr. 
Parker A. Small of NIMH. 

9. Studies of Protein Structure 

Work on the structure of fibrinogen and of acto- 
myosin has continued. Insight into the structure 
of fibrinogen has been obtained by coupling the 
molecule with a fluorescent dye : 1-dimethyl amino 
naphthalene-5-sulfonyl chloride. Studies of the 
degree of polarization of the fluorescent light from 
the complex in media of different viscosities leads 
to the conclusion that fibrinogen must contain sub- 
units even smaller than those liberated by trypsin 
digestion. Essentially what is found is that there 
must be a great deal of flexibility in the molecule, 
more than would be expected on the basis of pre- 
viously proposed subunit structures. The varia- 
tion in fluorescent intensity with pH suggests ty- 
rosine residues in the protein are involved in the 
complex formation. 

A new form of actomyosin has been isolated 
from rabbit muscle. Hitherto actomyosin has 
been formed only with the filamentous (F-form) 
of actin and this preparation is a very highly vis- 
cous one. The new form of actomyosin now iso- 
lated appears to contain the globular or monomeric 
form of actin (G-form). Preliminary studies 
suggest that in the intact muscle it may be the 
G-form of actomyosin that is normally present. 

10. Phospholipid Biosynthesis in Red Blood 

Cells 

Red blood cell membrances (ghosts) have been 
shown to incorporate labeled fatty acids into phos- 
pholipids. Most of the incorporation occurs in 
lecithin and essentially all of that in the /? posi- 
tion of the molecule. Evidently there is little or 
no de novo synthesis of lecithin but rather the 



labeled fatty acid is activated to the Co A deriva- 
tive and incorporated into lysolecithin. The 
source of the acceptor lysolecithin is not clear since 
phospholipase activity has not yet been demon- 
strated in the ghosts. Similar results were ob- 
tained using intact red blood cells. 

The above studies were carried out with red cell 
membrances obtained from two kinds of sheep. 
One variety has red cells characterized by a very 
high intracellular concentration of potassium 
while the other has red cells with a low intracellu- 
lar concentration of potassium. Since the incor- 
poration of labeled fatty acid into lecithin was 
comparable in the two kinds of red cells, it appears 
that the phospholipid turnover studied is not in- 
timately related to the mechanisms of potassium 
transport in these cells. 

11. Formation of Chylomicrons From Endo- 

genous Sources 

Dietary fat is absorbed primarily in the form 
of chylomicrons delivered to the blood stream by 
way of the thoracic duct. However, it is known 
that very low density lipoproteins can appear in 
the circulation during fasting. Studies were un- 
dertaken to characterize the lipoproteins of the 
chyle in animals on fat free diets. 

Rats on a fat free diet continued to produce 
chylomicron-like lipoproteins demonstrate in tho- 
racic duct chyle. The lipid in the chyle is pre- 
dominantly triglyceride and most of it is asso- 
ciated with particles of density less than 1.006. 
The chyle obtained directly from intestinal lym- 
phatics was similar to that obtained from the 
thoracic duct. It thus appears that the lipopro- 
teins arise in the intestine. This lipoprotein con- 
tinues to be produced even when only saline is 
given. It is suggested that endogenous substrates 
are utilized by the intestinal mucosa in order to 
form these "fasting" chylomicrons. 

12. Studies on Structure and Mechanism of 

Action of Parathyroid Hormone 

Dr. Gerald Auerbach of NIAMD has isolated 
and partially purified parathyroid hormone from 
phenol extracts of bovine parathyroid glands. 
Further purification has been achieved using 
Sephadex columns. Physical studies and end- 
group analysis show that the material, with a 



NATIONAL HEART INSTITUTE 



93 



potency of 3,000 U.S. P. Units per mg, is now pure. 
This material is being used for studies of the mech- 
anism of action of the hormone. 

Section on Chemistry 

The work of the past year may be summarized 
in four areas. These are (1) the development of 
gas phase chromatographic techniques for the 
qualitative and quantitative analysis of biolog- 
ically important substances, (2) investigation of 
the isolation, structure, properties and biogenesis 
of plant alkaloids, (3) studies of the components 
of the kallikrein-kallidinogen-kallidin system, and 
of the chemistry of human polypeptide vasodila- 
tors, and (4) consultative and informal collabora-: 
tion with various intramural research groups of 
the National Institutes of Health seeking the spe- 
cific knowledge and equipment of the Laboratory 
for application to their particular problems. 

(1) Gas Phase Chromatographic Methodology 

A method has been developed for the analysis of 
17-hydroxycorticoids in biological fluids. A sys- 
tem has also been developed for the analysis of 
nucleosides as their acetates. Hydrogen flame de- 
tectors have been evaluated for high molecular 
weight substances and found to be useful. The 
deleterious effect of copper and metallic columns 
and fittings has been confirmed. The preparative 
technique previously outlined has proved to be of 
great utility in several problems. A column pack- 
ing has been found (neopentyl glycol succinate) 
which is highly efficient for the separation of cho- 
lesterol and desmosterol. 

(£) Alkaloid Work 

Work has continued on minor alkaloids of Or- 
mosia panamen-sis using gas chromatographic 
techniques. Three new bases have been isolated, 
one being identical with a synthetic transforma- 
tion product of another. Several degradations 
were carried out on an alkaloid from Astroeasia 
phyllantoides but its structure is still unknown. 
Structural investigations on the three major alka- 
loids from the Ormosia species have been intensi- 
fied. The problem has been found to be peculiarly 
refractory. The alkaloids resist nearly all classi- 
cal degradations and it is felt that a new chemical 
linkage must be involved. 

The structures of Amaryllidaceae alkaloids, 
ambelline and amaryllisine have been elucidated, 



employing modern spectral methods. Biogenetic 
experiments on the Amaryllidaceae alkaloids have 
uncovered a branching point in the biosynthesis 
between phenylalanine and tyrosine. A cell-free 
enzyme system, capable of methylating norbella- 
dine to a known biogenetic intermediate has been 
discovered and partially purified. The cof actor is 
S-adenosylmethionine. 

Spectral studies have been completed on hydro- 
gen bonding phenomena in several types of com- 
pounds yielding detailed knowledge of the rota- 
tional configuration of hydroxyl groups in these 
natural materials. 

The structure of the alkaloid, tecomanine, has 
been elucidated. It is the third known example 
of a monoterpenoid cyclopentanoid alkaloid and is 
chemically related to the ant oil, iridiomyrmecin. 

(3) The Kallihrem-Kallidinogen-Kallidin 
System 

The human vasodilating substance kallikrein 
owes its action to the formation of the physiologi- 
cally active polypeptide kallidin. The latter com- 
pound is formed when kallikrein acts on kalli- 
dinogen, a component of human plasma. Work 
directed to the isolation of kallikrein, kallidin and 
kallidinogen has been carried on as a joint study 
with the Laboratory of Cardiovascular Physi- 
ology. 

Samples of human urinary, pancreatic and hog 
pancreatic kallikreins described in the last report 
have been purified (~90%) and characterized. 

Human plasma kallidinogen has been isolated 
in 28% yield and a 34-fold purification achieved. 

A new technique for the preparation and acti- 
vation of hydroxylapatite has been developed. 
With this adsorbent, kallidinogen has been sepa- 
rated into six components; the biological activity 
coinciding with one of them. 

{If) Miscellaneous Informal Collaborative 
Research 

In addition to conducting the research cited 
above, a significant amount of time and effort has 
been spent by several members of the laboratory 
to help other scientists of the National Institutes 
of Health with specific problems. Our help has 
been sought primarily in three areas: (1) Large 
scale processing of microorganisms, plant mate- 
rials, biological fluids, glands, and culture media. 
There has been an increased utilization of the 100- 



94 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



gallon ferment er for the growing of non- 
pathogenic microorganisms. As a result of the 
reorganization of the various laboratories, opera- 
tion of the large scale laboratory as a joint NHI- 
NIAMD project has been discontinued although 
most of the facilities will still be available to the 
NHI investigators. 

LABORATORY OF CLINICAL BIOCHEMISTRY 

Amine Biogenesis and Metabolism 

Work in this area has continued along several 
lines. Inhibitors of the enzyme, dopamine /J-oxi- 
dase, have been investigated in vitro and in vivo. 
All benzyloxyamines tested produced inhibition 
by a mechanism which indicates competition with 
substrate. However, when inhibitor is preincu- 
bated with enzyme before adding substrate, the 
inhibition becomes noncompetitive. Three pro- 
cedures for demonstrating the in vivo effects of 
these inhibitors have been utilized : (a) ability to 
block the conversion of dopamine to norepine- 
phrine in the guinea pig heart depleted of nor- 
epinephrine with aramine; (b) ability to block 
restoration of norepinephrine levels in guinea pig 
heart following administration of a short-acting 
depleting agent; (c) ability to inhibit the guinea 
pig adrenal enzyme. Benzyloxyamine has been 
shown by all three criteria to be active in vivo. 
However, the in vivo inhibition with single doses 
as large as 200 mg/Kg is no more than 80% and 
does not last for more than a few hours. The m 
vivo effects of other inhibitors are more variable. 
All compounds of this class produce hemolysis, 
methemoglobm, enlarged spleen, and convulsions. 
More compounds are being investigated and fur- 
ther attempts are being made to evaluate in vivo 
effects. 

Certain findings suggest the existence of self- 
regulatory mechanisms for norepinephrine syn- 
thesis. For example, conversion of administered 
dopamine to noradrenaline is negligible in normal 
animal tissues in vivo. Following depletion of 
norepinephrine stores with aramine or a-meta- 
tyrosine conversion is rapid and extensive. Iso- 
topic experiments have been used to corroborate 
this. Even without isotopes rapid repletion of 
depleted stores can be observed readily. The 
possibility that norepinephrine biosynthesis may 



be regulated by norepinephrine itself (as a feed- 
back mechanism) or by the action of some other 
humoral agent released by the norepinephrine is 
being investigated. 

It has been shown that labeled tyramine and 
norsynephrine (octopamine) can be converted to 
urinary epinephrine and normethanephrine. The 
significance of this alternative route remains to be 
determined. However, some of the pharmacologi- 
cal properties of tyramine (at high doses) may 
be due to conversion to norepinephrine. 

It has been possible to label the norepinephrine 
in heart, spleen, brain, as well as adrenal gland, 
by administering 100 microcuries of tyrosine- 
U-C 14 to individual guinea pigs. This has made 
it possible to evaluate the turnover of the hormone 
in the peripheral tissues. Initial studies have 
proven beyond question that the labeled material 
in the tissues is indeed norepinephrine. Prelimi- 
nary findings corroborate a half-life of several 
days for adrenal norepinephrine. By contrast, 
half-lives in other tissues are of the order of sev- 
eral hours. Additional animals are being carried 
through to obtain more precise data. The iso- 
lated perfused heart has been found to contain all 
the catalysts necessary to form noradrenaline from 
the dietary amino acid tyrosine. The radioactive 
hormone and its precursor, dopamine, have been 
identified by many procedures. Under the condi- 
tions employed as much as 0.05 ng of noradrenaline 
was formed per gram of heart per hour. The per- 
fused heart will be useful in future studies of cate- 
cholamine biochemistry. 

a-Methylamino acids, such as a-methyl-dopa and 
a-methyl 5-hydroxytryptophan, are excellent in- 
hibitors of the decarboxylation of the naturally oc- 
curring aromatic amino acids. Some controversy 
has existed concerning the mechanism of this in- 
hibition. It is now apparent that the kinetics of 
this inhibition vary with the experimental 
conditions employed. If the enzyme is preincu- 
bated with inhibitor before the addition of sub- 
strate the characteristics of the inhibition are 
those of a noncompetitive inhibitor, while if no 
preincubation is employed competitive inhibition 
kinetics are obtained. Pyridoxal phosphate, the 
coenzyme for this reaction, also plays an important 
role. It protects the enzyme against the inactiva- 
tion which occurs during preincubation, as well 
as against the effect of the a-methylamino acids. 



NATIONAL HEART INSTITUTE 



95 



Once the enzyme has been inhibited, however, it 
has not been possible to reverse this inhibition by 
addition of coenzyme. 

Studies with rat liver phenylalanine hydroxyl- 
ase have shown that this enzyme can also convert 
tryptophan to 5-hydroxytriptophan (5HTP). 
The physiological significance of this reaction in 
the formation of serotonin has been under investi- 
gation. Inhibition of the enzyme in vivo causes 
little if any effect on serotonin levels in the tissues. 
This finding coupled with the distribution of the 
enzyme (fomid only in liver) makes it apparent 
that phenylalanine hydroxylase is not responsible 
for serotonin f ormation in vivo. Attempts to show 
the enzymatic conversion of tryptophan to 5HTP 
in tissues other than liver have not been successful. 

The inhibition of monoamine oxidase (MAO) 
by iproniazid, labeled in the isopropyl moiety, has 
been investigated. It appeal's that isopropyl hy- 
drazine is formed nonenzymatically by an oxida- 
tive cleavage of the iproniazid molecule. This 
volatile hydrazine appears to be an intermediate 
in the formation of the true inhibitor from 
iproniazid. Studies of the binding of inhibitor 
to enzyme have been performed, with emphases 
on the kinetics and type of inhibition. Attempts 
to solubilize or show a lipid requirement for mono- 
amine oxidase have not succeeded. 

Collagen and Hydroxyproline 

Studies on the formation of collagen and hy- 
droxyproline in cell-free systems have been con- 
tinued. It has been possible to determine some 
requirements of the chick embryo ribosomal sys- 
tem and to show an RNA involvement. Attempts 
are now being made to determine the exact stage 
at which hydroxylation of proline occurs : whether 
it is at the stage of prolyladenylate, S-RNA pro- 
line, or a later intermediate. It has been found 
that the oxygen involved in the conversion of 
proline to hydroxyproline is derived from 2 18 and 
not H 2 18 . Thus the enzyme must be an oxygenase 
and the reaction for hydroxylation of proline 
would be written as : 



H 



-COOH 



II 



C— 0— R 



l/20j 



H 



C— O— R 



Collagen OH Pr<- 



R=adenylate, S — RNA, or other intermediate activated form 



Proteins and Peptides 

Studies have continued on the biosynthesis of 
the chromopeptide antibiotic actinomycin. Radio- 
isotope experiments on the precursors of the ac- 
tinomycin molecule have revealed that L-valine 
but not D-valine is used for synthesis of the D- 
valine in actinomycin. Evidence obtained with 
L-methionine-C 14 H 3 and L-valine-1-C 14 and gly- 
cine-!.- and -2-C 14 have indicated that in vivo, a 
direct methylation of L-valine and glycine is re- 
sponsible for the synthesis of N-methyl-L-valine 
and sarcosine, respectively. A rapid method of 
assay for actinomycin, based on the selective solu- 
bility of the antibiotic in organic solvents has 
been developed. It was possible to demonstrate 
with the use of the assay procedure and radio- 
isotopes that the incorporation of a precursor into 
actinomycin is generally a rapid, efficient and 
linear process. With inhibitors of protein syn- 
thesis such as chloramphenicol, puromycin or 
ethionine it was possible to show an inhibition of 
protein synthesis by Streptomyces antibioticus (as 
measured by incorporation of a C 14 labeled amino 
acid) with a simultaneous stimulation (2- to 3- 
fold) of actinomycin synthesis. These results 
indicate that the mechanism of synthesis of the 
peptide antibiotic, actinomycin, differs from that 
postulated for protein synthesis. 

Brain can actively synthesize y-glutamylhisti- 
dine from histidine and a y-glutamyl donor such 
as glutathione. The enzyme which catalyzes this 
reaction is y-glutamyltranspeptidase. Up to this 
time the presence of this enzyme in brain and the 
ability of histidine to act as an acceptor of the 
y-glutamyl group have not been ascertained. Un- 
equivocal demonstration of the enzyme in brain in 
the present study was possible because of the de- 
velopment of a highly sensitive and specific fluoro- 
metric method of assay. That y-glutamylhistidine 
is formed in vivo is suggested by its presence in 
human urine. It has not previously been demon- 
strated in animal tissues. Numerous y-glutamyl 
derivatives have been isolated from plant tissues 
and certain microorganisms. Recently, additional 
y-glutamyl and /3-aspartyl peptides have been iden- 
tified in human urine by another laboratory. 
Since these peptides were also found in the urine 
of fasting individuals their endogenous origin is 
strongly implicated but their immediate source is 



96 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



still unknown. A possible source of y-glutamyl 
and /?-aspartyl peptides in urine is collagen. In 
keeping with this hypothesis was the finding, in 
humans, of a significant increase (at least 5-fold) 
in the urinary excretion of /J-aspartyl histidine 
following an increase in dietary collagen and 
gelatin. This finding is of great interest and the 
presence in collagen of other /?-aspartyl peptides 
such as y8-aspartyl glycine and sei'ine are currently 
under investigation. In line with this work, in 
vitro digestion of collagen with proteolytic en- 
zymes is also under study. If such digests contain 
/8-aspartyl and y-glutamyl peptides (as others have 
suggested) then this will be strong evidence for 
the occurrence of these unusual linkages in this 
protein. 

Studies have been carried out on the distribution 
of the dipepticles carnosine and homocarnosine. 
Only the brain was found to contain them, the 
white matter of human brain having from 2 to 4 
times as much dipeptide as gray matter. This is 
just the reverse of the distribution of the precursor 
GABA, which is concentrated in the gray matter. 
Urine was found to contain homocarnosine with a 
daily excretion ranging from 2.0 to 9.8 ^moles per 
day. If it can be shown that the homocarnosine in 
urine does indeed come from the central nervous 
system and not from the diet, the level of this 
dipeptide in urine may serve as a useful index of 
brain metabolism. Concomitant with this work a 
systematic study was also made of the uptake of 
histidine, carnosine and homocarnosine by rat 
brain slices. Histidine and carnosine were accu- 
mulated by slices against a concentration gradient 
but homocarnosine was not. An examination of 
the slices after incubation revealed little or no 
metabolism of histidine or homocarnosine even 
after 3 hours of incubation. Carnosine, one the 
other hand, was hydrolyzed to histidine and /3- 
alanine. The disappearance of carnosine could be 
accounted for entirely from the amount of /3- 
alanine formed. It is interesting that sarcoma S- 
37 ascites cells could not take up carnosine or homo- 
carnosine even though they showed a marked 
activity with respect to histidine uptake. The up- 
take of carnosine by brain slices was studied 
further with regard to energy requirement, efflux, 
and inhibition. The latter study revealed that the 
best inhibitors were other peptides, especially /?- 
alanyl dipeptides. However, histidine and espe- 



cially /^-alanine were almost as effective inhibitors 
as were peptides. 

The conversion of phenylalanine to tyrosine is 
being used in an attempt to gain information re- 
garding the turnover of proteins in mammals and 
in bacteria. It was observed some time ago that 
an anomalous situation arose when one measured 
the specific activity of protein tyrosine after ad- 
ministering radioactive phenylalanine to a dog. 
The time course of decay of the tyrosine in plasma 
protein was different from that observed if tyro- 
sine itself were administered. The origin of this 
effect is under study using the purified blood pro- 
teins of dogs and various combinations of tritium- 
and carbon-labeled phenylalanine and tyrosine. A 
similar study is being attempted in bacteria using 
a strain of Pseudomonas in which hydroxylation 
of phenylalanine to tyrosine is an inducible 
enzyme. 

As part of a study on the over-all metabolism of 
proteins in the brain, a Ca ++ -stimulated, soluble 
neutral protease has been extensively purified. A 
stable, active enzyme has been prepared using 
(NH. 1 ) 2 S0 4 fractionation, isoelectric precipita- 
tion, and DEAE-cellulose chromatography. Us- 
ing casein as a substrate the characteristics of the 
enzyme have been determined. For maximal ac- 
tivity Ca^, EDTA, sulfhydryl, and buffer, pH 
7.4 are necessary in appropriate amounts. In 
search of the mechanism of action of the enzyme 
a series of model peptides was investigated with 
sensitive spectrophotometric methods. No action 
could be observed, indicating tentatively that the 
enzyme is unlike trypsin, chymotrypsin, carboxy- 
peptidase A or B, papain or known cathepsins in 
its mode of action. Since the enzyme acts on oxi- 
dized ribonuclease, this well-characterized pro- 
tein is being used in a study of the mode of action 
of the protease. End group analysis and finger 
printing techniques are being adapted for this 
purpose. 

Amino Acid Uptake by Animal Tissues 

A continuing study of the significance of aro- 
matic amino acid uptake by brain has been pur- 
sued. Recently, earlier findings regarding the up- 
take of tyrosine by rat brain in vivo have been 
extended to phenylalanine and tryptophan. A 
comparison between brain and other organs has 
served to emphasize that brain is unique in this 



NATIONAL HEART INSTITUTE 



97 



regard. Competitive relationships previously 
demonstrated have been used to develop an hy- 
pothesis concerning the possible mechanism of 
mental retardation hi conditions involving high 
blood levels of amino acids such as in phenylke- 
tonuria. Certain dfferences between amino acid 
uptake in vivo and in vitro led to suggestions about 
the anatomical location of portions of the uptake 
mechanism. Specifically, the marked stereo- 
specificity found in vivo but not in vitro is con- 
sidered to be a function of the "blood-brain bar- 
rier" or a related mechanism. To obtain verifica- 
tion of this hypothesis and to gain other informa- 
tion a study is in progress concerning the pene- 
tration of amino acids into the brain of newborn 
rats. This study is based on suggestions from 
other laboratories that newborn animals do not 
exhibit a barrier mechanism. 

A study of the amino acid uptake of the Sar- 
coma 37 ascites cell has also been undertaken with 
the aim of developing a model system for the 
study of the effects of certain antitumor agents. 
In the initial phases of this program the trans- 
port of amino acids in vitro was investigated. 
With certain minor differences the transport of 
amino acids resembled the well-described case of 
the Ehrlich ascites cell. In addition, in vitro 
levels of alkylating agents which prevented the 
growth of these cells when the cells were reinocu- 
lated into mice had no effect on the transport sys- 
tem. An unusual effect of the antimetabolite 
p-fluorophenylalanine was noted in these studies 
and is being pursued at present. Simultaneous 
exposure of the cells to tyrosine and o-fluoro- 
phenylalanine led to significant, reproducible in- 
creases in tyrosine uptake of the order of 25 to 
35%. Further, a much greater effect on the up- 
take of tryptophan, of the order of 200 to 300%, 
was noted. It has seemed appropriate to investi- 
gate this effect and to determine, if possible, its 
origin. Accordingly, labeled p-fluorophenylalan- 
ine is being employed to study the effect of amino 
acids on this compound and the conditions of the 
experiments are being altered to determine if this 
effect can be explained on the basis of counter flow 
or if some other explanation must be sought. 

Biosynthesis of Phospholipids and Other Nitro- 
gen Containing Lipids 

Toward the end of 1961 studies on the biogene- 
sis of phospholipids, which had been temporarily 



discontinued, were started again. During investi- 
gations on the incorporation of ethanolamine-C 14 
into phospholipid it was found that activity was 
greatest in microsomes fortified with fatty acids. 
However, the product formed on addition of pal- 
mitate and ethanolamine was found to be the cor- 
responding amide, palmitoyl-ethanolamide and 
not cephalin. In fact, all the ethanolamine was 
incorporated into this amide. The reaction has 
been shown to be a net synthesis rather than some 
backward reaction of a hydrolytic mechanism. 
The fatty acid amides of ethanolamine have been 
reported to have anti-inflammatory and antisero- 
tonin activities. The significance of these com- 
pounds and of the enzymes involved in their for- 
mation and metabolism are currently being 
investigated. 

Studies Relating to Vitamin B 12 

The conversion of vitamin B 12 to B 12 coenzyme 
is depicted in the following way : 



CN 

\ I / ATP, FMNH. 

CO 

/| \ SH- 



5'-deoxyadenosyl 

\l / 

CO 

/I \ 



+CN-+3(PO,) 



In an attempt fully to understand this unique 
reaction, the enzyme was purified from extracts of 
Clostridium tetpmomorplium. The reaction has 
been studied in a variety of ways, including (a) 
incorporation of the adenosyl moiety of ATP into 
coenzyme, (b) release of cyanide, (c) spectral 
changes, (d) P0 4 release from ATP, and (e) for- 
mation of enzymatically active coenzyme. It has 
not been possible to demonstrate a partial reac- 
tion, and the available data favor a concerted re- 
action, as presented in the above equation. The 
form hi which the phosphates from ATP are re- 
leased during coenzyme synthesis could not be 
studied until the high ATPase activity in the ex- 
tracts was reduced. Once achieved it was possible 
to show that inorganic tripolyphosphate is re- 
leased from ATP during the enzymatic reaction : 

Adenine-ribose-P-P-P— >5'deoxyadenosyl moiety 
+ tripolyphosphate 

The role of vitamin B i2 in one carbon transfer 
is also being investigated. The final reaction in 
methionine biosynthesis involves the transfer of 
the methyl group from CH 3 tetrahydrofolic acid 
(CH3THF) to homocysteine. Adenosylmethio- 



98 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



nine (AMe) and a B 12 coenzyme analogue 
(CH 3 B 12 ) have been implicated in this reaction. 
This conversion is being studied in both an animal 
system (pig liver) and in extracts of E. coli, using 
C»H 3 THF, methyl H 3 -Ame, and C 14 H 3 B 12 . The 
latter compound has been chemically synthesized. 
It is extremely light sensitive, similar to the B i2 
coenzyme, and during light inactivation the car- 
bon to cobalt bond is broken via homolytic cleav- 
age; CH 3 (35%) and CH 3 OH (percentage not 
yet determined) being formed. The development 
of simple assays has facilitated studies on methio- 
nine biosynthesis. It has now been demonstrated 
that AMe is necessary for the reaction from 
CH 3 THF in both systems studied, although the 
methyl group of AMe is not transferred to homo- 
cysteine. CH 3 B 12 is an absolute requirement in 
the bacterial system, not being replaced by vita- 
min B 12 or Bi 2 coenzyme. The CH 3 group of 
CH 3 B 12 is transferred during the course of the re- 
action, but AMe is not needed in this reaction. 
ATP and TPNH, which are needed along with 
AMe for the reaction from CH 3 THF, are not nec- 
essary for the reaction from CH 3 Bi 2 . Although 
CH 3 B 12 does transfer its CH 3 group, the evidence 
indicates that free CH 3 B 12 is not an intermediate 
in the over-all reaction. 

Development of Analytical Procedures 

A procedure has been developed for the assay 
of N-dimethylaminoguanine in the presence of 
guanine. The former is a trace component in 
S-RNA. The method should make possible the 
evaluation of its significance. 

A procedure for determination of phenethyl- 
amine in the urine of phenylketonurias has been 
developed. 

Since the last report, attention has been given to 
those compounds which have been difficult to 
study by gas chromatography. Two approaches 
have been taken. The first has been a search for 
a suitable polar liquid phase and the second, the 
preparation of derivatives likely to chromato- 
graph well. Efforts have been directed primarily 
to the 2,4-dinitrophenyl and phenylthiohydantoin 
derivatives of the hydroxy and basic amino acids. 
This has met with limited success but the work is 
continuing. 



Section on Biochemical Genetics 

RNA and the Genetic Code 

This research encompasses the following 
aspects: (1) development of a cell-free assay for 
messenger RNA, (2) synthesis of a natural pro- 
tein directed by purified preparations of messen- 
ger RNA, (3) investigating the characteristics of 
messenger RNA, and (4) the characteristics of 
the genetic code. 

Cell-Free Assay for Messenger RNA. Pro- 
tein synthesis was studied by following C 14 -amino 
acid incorporation in cell-free E. coli extracts. 
Techniques were developed which stabilized such 
extracts so that they could be stored frozen for 
long periods of time without undue loss of activ- 
ity. Small amounts of DNAase added to reaction 
mixtures were found to inhibit amino acid incor- 
poration. Thus, the genetic material appeared to 
be directing protein synthesis in this completely 
cell-free system. Further experiments suggested 
that protein synthesis ceased because endogenous 
messenger RNA had been depleted. Purified 
preparations of messenger RNA then were added 
to reaction mixtures and were found to direct 
amino acid incorporation into protein with high 
efficiency. Such RNA fractions proved to be a 
new requirement for cell-free protein synthesis. 
This technique afforded a highly sensitive, cell- 
free assay for messenger RNA and provided the 
rationale for all subsequent work. 

The Messenger Role of Viral RNA. A natu- 
rally occurring template RNA, tobacco mosaic 
virus RNA, was used to direct protein synthesis 
in this system. The protein synthesized under the 
direction of TMV-RNA was shown to have many 
characteristics of TMV-protein. Part of the pro- 
tein formed could be purified with authentic 
TMV-protein by repeated isoelectric precipitations 
and DEAE column chromatography. TMV- 
protein is an excellent one to characterize for its 
entire amino acid sequence is known. Digestion 
with trypsin converts it into 12 peptides of estab- 
lished amino acid composition and chromatog- 
raphic properties. Seventeen peptides were iso- 
lated after digesting with trypsin and each peptide 
was subjected to amino acid analysis and its radio- 
activity was counted. TMV-RNA was found to 



NATIONAL HEART INSTITUTE 



99 



direct C 14 -amino acids into expected peptides. 
Thus, the cell-free synthesis of this protein and the 
messenger role of viral RNA was demonstrated. 

Fate of Messenger RNA. Synthetic polynu- 
cleotides of known chemical constitution were 
assayed to see if they could direct the synthesis of 
simple proteins. Polyuridylic acid was found to 
stimulate greatly the incorporation of phenyl- 
alanine into protein. The product of the reaction 
was identified as polyphenylalanine and sRNA 
was shown to be an intermediate in its synthesis. 

The effect of secondary structure upon the tem- 
plate activity of poly U was also investigated. 
Poly U in solution occurs as randomly coiled, 
single-strands. In the presence of polyadenylic 
acid, double- and triple-stranded polynucleotides 
are formed. Such double- and triple-stranded 
polynucleotides were unable to direct the synthesis 
of polyphenylalanine. In addition, different 
preparations of randomly-ordered polynucleo- 
tides with varying degrees of secondary structure 
were assayed for template activity. Preparations 
with considerable secondary structure had little 
template activity. These results strongly suggest 
that single-strandedness is requisite for messenger 
RNA activity. Another factor was found which 
influences the messenger activity of RNA ; that is, 
its molecular weight. Poly U containing 300 or 
more uridylic acid residues had markedly higher 
template activity than shorter chains. 

Radioactive poly U was synthesized and the fate 
of messenger RNA in this system was followed. 
Addition of poly U resulted in a specific aggrega- 
tion of ribosomes which sedimented at 100-130 S. 
Protein synthesis occurred initially only upon 
heavy ribosomes. Thus, these ribosomal aggre- 
gates were shown to be the site of protein synthesis. 

Evidence was obtained which strongly suggested 
that poly U acted cataly tically in this system ; that 
is, one molecule of poly U directed the synthesis 
of more than one molecule of polyphenylalanine. 

Characteristics of the Genetic Code. Since 
the sequence of uridylic acid residues in poly U 
was the RNA codeword corresponding to phenyl- 
alanine, the use of synthetic polynucleotides 
afforded a relatively simple experimental means 
of determining the nucleotide compositions and 
characteristics of genetic coding units. Randomly- 
mixed polynucleotides of known base composition 
were synthesized and found to direct many other 

705-685—63 8 



amino acids into protein in a highly specific fash- 
ion. The nucleotide composition of coding units 
corresponding to almost all amino acids was deter- 
mined in this manner. Various characteristics of 
the code such as the coding ratio, degeneracy, spec- 
ificity, efficiency, universality of the code, etc., 
were investigated. Evidence was obtained sug- 
gesting that the coding ratio (the number of nu- 
cleotides per codeword) is three. A degenerate 
code is one in which two or more coding units code 
for one amino acid. The code was shown to be 
highly degenerate. However, code words were 
found to display striking specificity for their 
respective amino acids. Comparison of the nu- 
cleotide composition of the coding units with 
amino acid replacements occurring in mutant pro- 
teins suggested that a large portion of the code was 
universal ; that is, phylogenetically diverse species 
have similar genetic codes. Such experiments per- 
mit a detailed description of the nature of the 
code. 

CLINICAL ENDOCRINOLOGY BRANCH 

The work of the Clinical Endocrinology Branch 
has included chemical, physiologic and clinical 
studies related to the physiology of the adrenal 
cortex, physiologic and clinical studies related to 
phosphorus and calcium metabolism and to the 
function of the parathyroid glands, studies of 
renal mechanisms for excretion of salt and water, 
and studies not closely related to these. 

Studies of adrenal steroid metabolism have in- 
cluded the development of a method for determin- 
ing plasma androsterone and etiocholanolone, 
studies in the biogenesis of adrenal steroids in 
vitro by slices of rat and beef adrenals, studies on 
the role of the pituitary and of the renin- 
angiotensin system in control of adrenal steroid- 
ogenesis in the dog, in man and in the rat, studies 
on the effect of acute expansion of plasma volume 
on aldosterone secretion and excretion in subjects 
with primary and secondary aldosteronism and 
hypertension, and studies of the pathologic physi- 
ology of the kidneys and adrenal cortex in a new 
syndrome thus far observed in three patients. 

Adrenal Function 

In the course of studies in this Branch on 
adrenal physiology in general and on periodicity 
in adrenal function in particular, it was necessary 
to develop methods for measurement of 19-carbon 



100 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



adrenal steroids. A knowledge of behavior of 
such steroids is required to define the pattern of 
adrenal responsiveness to a series of agents known 
to influence steroidogenesis. It has been reported, 
furthermore, that periodic disease may be related 
to periodic increases in the secretion or decreases 
in the destruction of 19-carbon steroids. Samples 
of androsterone and etiocholanolone with high 
specific activity were obtained by the Wilzbach 
method. The steroids were separated and puri- 
fied by a series of chromatographic steps of which 
a passage on thin layer proved essential to elimi- 
nate the "blank" susceptible to acetylation. Un- 
known steroids were extracted from blood and 
labeled, together with H 3 labeled tracer, with 
Carbon 14. With subsequent chromatography, 
methods were developed to measure C 19 steroids 
in blood. In current studies, normal ranges and 
circadian fluctuations are being determined. 
Methods for steroid analysis involving gas chro- 
matography were further developed and patterns 
for several products of intermediary metabolism 
of steroids were determined. In cooperation with 
the Technical Development Branch, the gas chro- 
matographic method was extended to include dou- 
ble labeling with collection of effluent material 
after the writing of the gas chromatogram and 
subsequent analysis of steroids by the double iso- 
tope derivative method. 

Studies in the biogenesis of adrenal steroids by 
slices and sections of glands in vitro were con- 
tinued. The ascorbic acid depletion test of Sayers 
and Sayers was used to distinguish ACTH from 
renin, in view of the known presence of both sub- 
stances in, and their relative ease of extraction 
from renal tissue, and in view of the property of 
both to stimulate secretion of aldosterone, corti- 
costerone and Cortisol. It was found that whereas 
ACTH in single or sustained doses readily de- 
pletes ascorbic acid from the adrenal cortex of the 
hypophysectomized rat, angiotensin has no such 
property when given in either way. 

Sections of beef adrenal cortex were shown to 
secrete aldosterone, corticosterone and Cortisol in 
vitro. Secretion was gently stimulated by the 
addition to the medium of steroid precursors 
which appear beyond cholesterol in biosynthetic 
pathways. ACTH and angiotensin stimulate 
steroidogenesis from precursors preceding and in- 
cluding cholesterol. The results thus strongly 
suggest that ACTH and angiotensin act at or 



near a biosynthetic step involving cholesterol and 
that they act in a similar fashion. These studies 
have been extended to indicate stimulation of 
steroidogenesis by 3' 5' cyclic adenosine mono- 
phosphate. Current studies are designed to de- 
termine the similarity and differences between the 
stimuli provided by cyclic AMP and angiotensin 
or ACTH. Attempts to obtain similar results 
with rat adrenal sections continued to give equiv- 
ocal results. Rat renin appeared to be entirely 
ineffective in stimulating steroidogenesis by rat 
adrenal cortex even in the presence of rat serum, 
presumably containing renin substrate. Factors 
influencing steroidogenesis by the adrenal cortex 
were studied extensively in the dog. The role of 
acute surgery was re-investigated by comparing 
studies done one or two hours after surgery with 
similar studies performed at least 18 hours follow- 
ing surgical intervention. In this manner it was 
shown that neither hypophysectomy nor nephrec- 
tomy lowered aldosterone secretion when stud- 
ied in the resting conscious animal at a suitable 
interval after operation. The effects of synthetic 
ACTH were studied in hypophysectomized, 
nephrectomized dogs. Doses of synthetic ACTH 
with very marked effect on secretion of corticos- 
terone and of Cortisol had equivocal effects on the 
secretion of aldosterone. The effect of the renin- 
angiotensin system was further studied and dose 
response curves were prepared. At all doses at 
which angiotensin stimulated secretion of aldo- 
sterone, it could be shown also to stimulate secre- 
tion of corticosterone and of Cortisol. The effect 
of constriction of the renal artery, presumably a 
stimulus to endogenous renin secretion, was stud- 
ied by measurements of steroid secretion and of 
blood and lymph renin and angiotensin. Con- 
striction of the renal artery regularly induced 
hypertension and increased secretion of aldoste- 
rone, hydrocortisone and of corticosterone. 
Whereas in some animals it produced transient 
increase in the secretion of renin, the latter re- 
sponse was not consistently present. The effect of 
low salt diet in stimulating steroidogenesis was 
studied further. Extracts of kidneys from dogs 
deprived of sodium regularly induced hyperten- 
sion and stimulated steroidogenesis (aldosterone, 
corticosterone, Cortisol) in hypophysectomized, 
nephrectomized recipients, whereas extracts from 
salt loaded dogs had no effect. The effect of con- 
striction of the inferior vena cava on steroido- 



NATIONAL HEART INSTITUTE 



101 



genesis and on production of renin was examined. 
Secretion of renin into thoracic duct lymph could 
be regularly stimulated by caval constriction in 
salt depleted animals and this response was elim- 
inated by nephrectomy. It does appear that se- 
cretion of renin is one means by which caval con- 
striction induces increased steroid secretion. In 
these studies caval constriction had little influence 
on the secretion of corticosterone or Cortisol, but 
critical evidence on this point could be derived 
only in the absence of the pituitary. The effect 
of carboline derivatives reported to stimulate se- 
cretion of aldosterone was studied in hypophysec- 
tomized, nephi^ectomized and in hypophysecto- 
mized, nephrectomized decerebrate dogs with 
negative results. The results of physiologic studies 
in the dog indicate that the renin-angiotensin sys- 
tem can stimulate steroidogenesis by the adrenal 
cortex. Furthermore, the stimulus of hemorrhage 
requires the presence of the kidneys for maximum 
efficacy. It is possible that the kidneys participate 
in the stimulus by releasing renin but this is not 
clearly established and other mechanisms are not 
excluded. 

Clinical studies on the control of aldosterone se- 
cretion in primary and secondary aldosteronism 
and on the role of the adrenal cortex in hyperten- 
sion were continued. The results of acute expan- 
sion of intravascular volume, of sodium depriva- 
tion and of pharmacologic agents which block the 
action of aldosterone were observed in a number 
of additional subjects and compared to those in 
patients with essential or malignant hypertension. 
The patients with hypertension were also evalu- 
ated by determination of renin in the renal vein 
by catheterization and by the radioactive reno- 
gram. Expansion of plasma volume with salt- 
poor human serum albumin continued to prove ef- 
fective in distinguishing the patient with second- 
ary aldosteronism (e.g., in cirrhosis), in whom 
aldosterone secretion and excretion are markedly 
diminished by the procedure, from the patient 
with primary aldosteronism (e.g., aldosteroma), 
in whom no change of aldosterone secretion or ex- 
cretion is seen. In current studies the effect of this 
procedure on patients with unilateral renal dis- 
ease, hypersecretion of renin, hypertension and 
aldosteronism is being studied. Observations in- 
clude measurements of circadian fluctuation in al- 
dosterone excretion and an attempt to develop a 
method for measurement of circadian changes in 



aldosterone secretion. It has thus been shown 
that, contrary to published reports, the magnitude 
of circadian fluctuations in aldosterone excretion 
is not diminished by keeping the patients in bed, 
although the timing of the peaks and troughs may 
be changed. Patients with primary aldosteronism 
from adrenal tumor continued to show marked 
circadian fluctuations in aldosterone excretion, al- 
beit with a time pattern different from that of nor- 
mal subjects. In a series of normal subjects, 
"maps" of circadian fluctuation of aldosterone ex- 
cretion were greatly extended. As the pattern 
proved highly consistent from subject to subject 
(with high values in the morning and low values 
in the evening) , addition of cases greatly increased 
the reliability of the estimate. 

The relationship of aldosterone secretion to the 
juxtaglomerular apparatus and thus to renin and 
angiotensin secretion was further studied in three 
patients with a new syndrome. This syndrome 
was characterized by normal blood pressure, 
marked hypertrophy and hyperplasia of the jux- 
taglomerular apparatus, increased quantities of 
circulating angiotensin and marked increases in 
aldosterone secretion. The normal blood pressure 
and the hypersecretion of aldosterone persist de- 
spite expansion of intravascular volume with al- 
bumin. The hypokalemic alkalosis characteristic 
of the syndrome was readily reversible with al- 
dosterone antagonists, thus indicating an aldoste- 
rone- dependent renal origin for this manifestation 
of the syndrome. The blood pressure response to 
infusion of synthetic angiotensin showed marked 
resistance to the pressor activity of angiotensin in 
all subjects. Two of the patients received human 
renin and its effects were compared with those in 
a normal subject. Both showed not only a marked 
resistance to the pressor effects of human renin 
but also a failure of aldosterone secretion to in- 
crease with renin. In a normal subject, in con- 
trast, aldosterone secretion showed a graded in- 
crease with increasing doses of human renin. 

Calcium and Phosphorus Metabolism 

Studies of phosphorus and calcium metabolism 
and on the function of parathyroid glands in- 
cluded measurements of the effect of phosphate 
loading and of parathyroidectomy on renal tubu- 
lar transport of phosphate, studies of the effect of 
parathyroid extract on bone in vitro, on the rachit- 
ic animal in vivo, and on electrolyte and hydroxy- 



102 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



proline excretion in man, studies of tests designed 
to detect hyperparathyroidism and to correlate 
clinical, chemical and histological findings in this 
condition, and studies on the metabolic bone dis- 
eases of osteoporosis, sarcoidosis and renal failure. 

Phosphate loading over prolonged periods of 
time was found to produce decreases in tubular re- 
absorption of phosphate, albeit never to a figure 
which would indicate phosphate secretion. In all 
studies there was a fall of filtration rate and the 
role of changes in filtration rate was independently 
studied. By direct comparison of maximal phos- 
phate transport determined by increasing the 
serum phosphorus with that determined by in- 
creasing the filtration rate it was shown that filtra- 
tion rate was slightly but significantly correlated 
with maximal phosphate transport. The decrease 
in transport observed with phosphate loading was 
significantly greater than that expected from the 
fall of filtration rate alone. The effect of para- 
thyroid extract on renal electrolyte excretion was 
studied in parathyroidectomized dogs. The in- 
crease of phosphate excretion by decrease of reab- 
sorption was regularly associated with a rise of 
urinary pH. 

In vitro parathyroid extract was shown to in- 
crease the calcium content of the media surround- 
ing slices of weanling rat calvarium and studies 
were extended in an attempt to establish a concen- 
tration-response relationship for this effect. 
Treatment of rats with vitamin D induced a simi- 
lar increase in calcium in the medium when the 
calvaria were incubated in vitro. These studies 
were compared with similar ones in vitamin D de- 
ficiency where the opposite occurred; the studies 
are being extended to allow better evaluation of 
the role of parathyroid hormone and vitamin D 
in bone metabolism. Studies were carried out in 
puppies with rickets to elucidate the mechanism 
responsible for the hypophosphatemia character- 
istic of that condition. Preliminary results sug- 
gest that parathyroidectomy leads to rapid in- 
crease in serum phosphorus and in tubular reab- 
sorption of phosphorus, results which suggest that 
secondary over-activity of the parathyroid and 
not a defect of the renal tubule is responsible for 
the hypophosphatemia of rickets. The effect of 
parathyroid extract was measured in normal 
human subjects and in human patients with hypo- 
parathyroidism and special attention given to the 
balances of calcium and magnesium and to the 



urinary excretion of hydroxyproline. Parathy- 
roid extract regularly increases urinary hydroxy- 
proline and this was taken as an index of destruc- 
tion of bone matrix as an early effect of parathy- 
roid hormone. Urinary calcium and magnesium 
decreased initially and later increased with the 
extract. The findings suggest an early direct tu- 
bular effect of extract on calcium and magnesium 
with later hypercalciuria and hypermagnesuria 
resulting from elevated serum levels. 

Studies designed to clarify the diagnosis of hy- 
perparathyroidism were extended. The effect of 
a standard infusion of calcium on 24-hour phos- 
phorus clearance was examined in a number of 
patients. The phosphorus clearance under basal 
conditions was compared with that found the fol- 
lowing morning after a calcium infusion given by 
night. Both the newer and the older type of test 
gave essentially the same information ; on the basis 
of positive results in these tests together with per- 
sistent hypercalciuria not responding to carbohy- 
drate-active steroids, a number of subjects with 
minimal criteria for hyperparathyroidism were 
surgically explored. Histologic lesions of the par- 
athyroids were found. 

The present studies also include measurements 
of circadian fluctuations of urinary phosphate ex- 
cretion over a thirty-hour period in normal men 
and women and in patients suspected of having 
diseases of the parathyroid. The normals show a 
highly repreducible pattern of phosphate excre- 
tion with relative retention during daylight hours 
and two peaks of relative phosphate loss during 
the night. Patients with hyper- and hypopara- 
thyroidism are being studied in a similar fashion 
and their curves compared with those of the nor- 
mal subjects. All the clinical, biochemical and 
histologic findings in patients explored for hyper- 
parathyroidism have been reviewed and compared. 
A disorder, not previously defined, involving 
abnormalities of calcium and of phosphorus 
excretion and histological hyperplasia of the para- 
thyroids without gross enlargement, was encoun- 
tered in a number of patients. In addition, a 
patient with hyperparathyroidism resulting from 
lipomyoadenoma of the parathyroid glands was 
studied before and after operation. This lesion 
has not heretofore been reported. In several pa- 
tients surgical intervention with partial removal 
of hyperplastic gland produced striking decrease 
in the rate of renal stone formation. Studies in 



NATIONAL HEART INSTITUTE 



103 



the metabolic bone diseases of idiopathic osteo- 
porosis, sarcoidosis and glomerular insufficiency 
included measurements of calcium absorption with 
standard oral doses of calcium 47 measurements of 
bone formation with standard intravenous doses 
of calcium 47 and metabolic studies in which the 
effect of various agents were tested. The patients 
with idiopathic osteoporosis were studied for a 
reported effect of 6a fluorotriamcinolone in favor- 
ing calcium retention. Whereas the agent did not 
produce calcium retention it did indeed appear to 
produce less loss of calcium than that produced by 
a comparable dose of other carbohydrate- active 
steroids with different chemical configuration. 
Patients with sarcoidosis were shown by isotope 
studies to have marked increase in calcium absorp- 
tion as originally detected by balance studies. It 
was found also that at the time they are suffering 
from hypercalcemia they show marked increases 
in the rate of bone formation. The increased ab- 
sorption of calcium could be decreased with 
carbohydrate-active steroids such as prednisone 
and there is a suggestion from early results that 
the increased formation rate may likewise respond 
to the steroids. The disorder of calcium metab- 
olism with glomerular insufficiency was further 
studied. Marked diminution in calcium absorp- 
tion followed the onset of acute nephritis and was 
regularly found with chronic glomerular insuffi- 
ciency. With the chronic disease, renal osteitis 
fibrosa was demonstrated byX-ray, by biochemical 
changes and by biopsy before treatment with vita- 
min D. With vitamin D the defect in calcium 
absorption was markedly improved or abolished 
and concomitantly the bone disease could be 
healed. Further studies are in progress to define 
the nature of the defect in calcium absorption with 
renal failure and to determine whether this bears 
any relationship to the remarkably high product 
of calcium by phosphate ions sustained in the 
serum of such patients. 

Studies on renal mechanisms for excretion of 
salt and water have included measurements of the 
role of adrenal steroids on urinary concentration 
and dilution, studies on the role of the adrenergic 
nervous system on sodium and water metabolism, 
studies of circadian fluctuation in salt and water 
excretion in normal and in abnormal subjects with 
special reference to the effect of posture, and stu- 
dies on the sodium metabolism of the fasting state. 
The role of adrenal steroids in promoting dilution 



and concentration of the urine was studied in pa- 
tients with Addison's disease from whom treat- 
ment was temporarily withdrawn and in normal 
subjects receiving steroid therapy. It was found 
that the antidiuresis of untreated Addison's dis- 
ease could be largely prevented by expansion of 
extracellular fluid volume without addition of 
steroid therapy. The expansion did not require 
the sodium ion, as expansion with albumin alone 
produced the same restoration of relatively nor- 
mal diluting power as that induced by saline. Pa- 
tients with Addison's disease were shown to have 
limited ability to produce concentrated urine and 
limited ability to reabsorb water under maximum 
antidiuretic hormone activity. The ability to con- 
centrate and to reabsorb water against a concen- 
tration gradient could be restored with desoxy- 
corticosterone in small doses or by simple dehy- 
dration, which presumably increased medullary 
solute concentration. Whereas maximal concen- 
tration of the urine could be shown with small 
doses of desoxycorticosterone, large doses did not 
restore concentrating power, a finding suggesting 
a role of desoxycorticosterone in promoting proxi- 
mal tubular sodium reabsorption. The role of the 
adrenergic nervous system in retention or excre- 
tion of sodium and water was studied in view of 
indirect evidence that the sodium retention for car- 
diac failure may depend in part upon excess of 
catecholamines. In long term studies, the effect 
of standard doses of desoxycorticosterone on so- 
dium balance were compared in the same patient 
on the same regimen with and without treatment 
with guanethidine at dosages which produced hy- 
potension and thus presumably peripheral cate- 
cholamine depletion. The effects of small doses 
of norepinephrine were measured directly in nor- 
mal subjects and compared with the effects of 
slightly larger closes. Results suggest that guane- 
thidine diminishes the sodium retention result- 
ing from a given close of desoxycorticosterone and 
thus suggest that catecholamines are indeed in- 
volved in the pathological sodium retention of 
some patients with edema. Small doses of nore- 
pinephrine induced retention of sodium and water 
in normal subjects, a finding which supports the 
above conclusions. In large doses, norepinephrine 
induced excretion of sodium and water. Studies 
are in progress to define better the possible role 
of catecholamines in the states characterized by 






104 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sodium retention. Studies on circadian rhythms in 
secretion of salt and water included extensive 
transverse mapping of urinary sodium, potassium, 
phosphate, hydrogen ion (titratable acidity and 
ammonium), aldosterone, 17 ketosteroids and 
Porter-Silber steroids over 30-hour periods dur- 
ing which collections were made at 3-hour inter- 
vals in groups of normal subjects large enough to 
establish statistical significance for the fluctua- 
tions observed. Results of these and of longitu- 
dinal studies of body temperature over protracted 
periods were subjects to thermal variance spec- 
trum analysis to evaluate the extent of fluctuations 
with circadian periods and that with other peri- 
odicities. Studies have been begun in which simi- 
lar results are obtained from patients with dis- 
ease. If the disease involves desynchronization 
of one body function from another, or if it involves 
a change of external timing of adrenal periodicity, 
it should be apparent from results of such analysis. 
In a patient with cancer and marked edema, for 
example, circadian periodicity of body tempera- 
ture was completely lost despite absence of fever 
at any time. 

Studies on the abnormal sodium metabolism of 
the fasting state were extended. The observation 
that the fasting subject may lose much more sodi- 
um that he loses when deprived of dietary sodium 
has been amply confirmed. It was shown that the 
extent of sodium loss appeared to be related to the 
degree of acidosis, and that a single patient might 
show wide differences in the extent of change of 
both these variables during different fasting 
periods. It was also shown that the degree of 
sodium loss was roughly correlated with the ex- 
tent of increase of urinary ketone bodies and that 
it could be prevented by feeding of carbohydrate 
alone. Further studies defining the role of keto- 
acidosis in this abnormal sodium loss of the fast- 
ing state are in progress. 

Studies not included in the above categories in- 
clude investigations into the mechanism of the de- 
velopment of atherosclerosis by a study of the rate 
of passage of large molecules across the aortic en- 
dothelium. It was shown that labelled lipopro- 
teins enter the arterial wall and that there are 
similarities in the dynamics of their passage into 
the wall and that of albumin and cholesterol 
shown for previous studies. It was clear that 
blood pressure was a prominent factor in deter- 
mining the rate of transport and that stretching 



of the wall was essential for this effect. Current 
studies with lipoprotein include an attempt to 
avoid loss of label from a given molecule. It is 
anticipated that the role of hormones or of hor- 
mone deficiencies in promoting atherosclerosis can 
be further defined with this technique. 

EXPERIMENTAL THERAPEUTICS BRANCH 

Biochemistry of Aromatic Amines 

Tyramine, a normal constituent of human urine 
and a vasoactive compound whose pressor effects 
are mediated by norepinephrine release, has been 
isolated and identified in mammalian tissues. 
With some exceptions, its occurrence in various 
species is confined to the central nervous system, 
the highest levels being in spinal cord (up to 5 
fig/gm), brain stem and cerebellum. Preliminary 
experiments suggest roles for the amine in modu- 
lating synaptic reflexes in the spinal cord and in- 
volvement in central responses to certain drugs. 
Also, the possibility that it may serve as a natural 
precursor of norepinephrine is under study. 

Sympathetic nerve and adrenal medullary tis- 
sues are known to be capable of synthesizing nor- 
epinephrine from its dietary precursor, tyrosine. 
This has not been shown previously for heart; 
indeed, there is a body of opinion that cardiac 
norepinephrine may be extracted from the circu- 
lating blood. Studies using isolated guinea pig- 
hearts (Langendorff procedures) have shown syn- 
thesis of C 14 -norepinephrine from C 14 -tyrosine 
in the perfusing fluid. This finding indicates that 
extensive evaluation of factors affecting synthesis 
and metabolism of the amine in heart will now be 
feasible. 

The final step in the synthesis of norepinephrine, 
the /3-hydroxylation of dopamine, is under inves- 
tigation in intact man. The impetus for develop- 
ment of sensitive techniques for quantifying 
clopamine-/J-oxidase activity in man is the prospect 
that inhibitors of this enzyme will soon become 
available for clinical studies. The urinary excre- 
tion of norepinephrine and its metabolites may 
afford a useful, albeit rather insensitive, index. 
The basis of a more sensitive test has been devel- 
oped with the finding that 4 to 9 percent of an 
oral dose of an artificial substrate of the enzyme, 
p-hydroxy-amphetamine (Paredrine), is excreted 
in the urine as its /?-hydroxylated metabolite, 



NATIONAL HEART INSTITUTE 



105 



p-hydroxynorephedrine. It is of interest that an 
adrenalectomized patient showed one of the high- 
est /J-hydroxylating activities. 

A veritable spectrum of catecholamine bio- 
chemistry was revealed by studies on the urine 
of 21 children with malignant tumors of the sym- 
pathetic nervous system. Elevated amounts of 
the following compounds were found : dopa, dop- 
amine, norepinephrine, normetanephrine, and 
vanilmandelic acid. Tyramine levels were not 
increased. A hitherto overlooked aspect of nor- 
epinephrine metabolism is suggested by the find- 
ing that, while 95 percent of an intravenous dose 
of H 3 -norepinephrine is excreted within 3 days 
as others have reported, the remaining radioac- 
tivity is excreted more slowly with a half time of 
about 8 days. 

The carcinoid syndrome, with its metabolic and 
pathologic implications, is still receiving major 
attention. It has been shown that many sym- 
pathomimetic amines induce typical flushing in 
carcinoid patients, the most potent agents being 
epinephrine and isoproterenol. Flush induction 
with minute doses of these compounds given in- 
travenously affords a useful diagnostic test at the 
bedside. Variants of the syndrome studied this 
year include : ( 1 ) a patient with metastases from 
a bronchial primary and with severe flushes, 
slightly elevated 5HIAA and normal blood sero- 
tonin. The flushes were controlled with predni- 
sone. A kinin-forming system has been demon- 
strated hi the plasma during flushes. (2) Another 
ipatient wtih metastatic gastric carcinoid with 
excretion of 5-hydroxytryptophan and large 
amounts of histamine in the urine. 

The exact pathogenesis of carcinoid endocardial 
fibrosis remains an enigma though a relationship 
to excess serotonin appears likely. A somewhat 
similar pathologic situation is the so-called endo- 
myocardial fibrosis (EMF) of Uganda. While 
the distribution of lesions differs from that in 
carcinoid, in both cases they are composed pre- 
dominantly of fibrous, rather than elastic tissue. 
Pertinent in this connection may be the fact that 
bananas, a fruit which we have shown contains 
large amounts of serotonin, are a dietary staple of 
the African in Uganda. The epidemiologic cor- 
relation between EMF and banana intake was con- 
firmed during a visit to Africa where discussions 
were held with personnel at the Makerere Medical 
College in Kampala with a view toward collabora- 



tive studies. We have shown hi normal volunteers 
that the fate of serotonin taken orally is the same, 
whether contained in bananas or capsules, with 
95 percent appearing in the urine as 5-hydroxy- 
indoleacetic acid. A 4-6-fold increase in platelet 
serotonin levels was observed after 2 weeks' treat- 
ment with oral serotonin, 160 mg/day. On stop- 
ping treatment, platelet serotonin returned toward 
normal logarithmically, with a half life of about 
3 days. This rather simple method of labelling 
platelets and measuring their turnover rate is 
being applied to study of platelet disorders. 

The cause of elevated plasma tyrosine levels in 
thyrotoxicosis appears to be complex. The effect 
of thyroid hormone on tyrosine-a-ketoglutarate 
transaminase activity in rat tissues is to increase 
levels of the enzyme which would tend to decrease 
rather than increase tyrosine levels. 

Methods for measuring urinary phenethylamine 
have been improved and observation of increased 
levels in phenylketonurics confirmed and ex- 
tended. Since this metabolite might be a more 
sensitive index of impaired hydroxylation of 
phenylalanine than blood levels of the amino acid, 
it is planned to study formation of the amine in 
heterozygotes during phenylalanine loading. 

Chemical Pharmacology and Therapeutics 

An interaction between indoles and catechol 
compounds is suggested by such phenomena as 
flush induction with catecholamines in carcinoid 
patients and coexistence of serotonin and norepi- 
nephrine in certain areas of the brain. Because of 
this, naturally-occurring and recently synthesized 
indole compounds were studied for effects on tissue 
stores of norepinephrine in laboratory animals. 
Serotonin, a-methyl-serotonin, a-methyl-trypta- 
mine and a-methyl-5-hydroxytryptophan were 
found to reduce the levels of norepinephrine in 
rat heart or brain, or both. In addition, a series 
of indole-phenylpiperazines were found to have 
similar activity. One of these, oxypertine, was 
studied in detail. It was found to decrease nor- 
epinephrine hi rat heart and brain without affect- 
ing brain serotonin. Catecholamine levels returned 
to normal within 8-12 hours after injecting the 
drug suggesting a rapid turnover of the amine. 
The compound also has sedative properties and is 
an effective blocker of a and /? adrenergic recep- 
tors, as defined by Allquist. In preliminary stud- 
ies, the compound has been shown to be highly 






106 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



effective in abolishing cardiac arrhythmias pro- 
duced by chloroform-epinephrine as well as by 
coronary occlusion. 

We are currently engaged in a long term, control- 
led study of the effectiveness of a-methyl-dopa 
( Aldomet) in 53 patients with severe hypertension. 
The drug is also being used frequently in hos- 
pitalized patients and has greatly simplified the 
management of many cases of malignant hyper- 
tension. In most patients (two-thirds) , the drug 
is more effective than other potent antihypertensive 
agents though optimal effect usually requires con- 
comitant use of a thiazide diuretic. Smoothness 
of effect, relative freedom from side effects, tran- 
quilizing properties and frequent control of blood 
pressure in recumbent as well as standing positions 
make Aldomet the current drug of choice for many 
hypertensive patients. 

Studies on the metabolism and mechanism of 
action of Aldomet have continued and permit the 
following conclusions: (1) The hypotensive effect 
is unrelated to the decarboxylase-inhibiting prop- 
erties of the compound. (2) The drug exerts its 
action by being decarboxylated to a-methyl-dop- 
amine which may in turn be /?-hydroxylated to a- 
methyl-norepinephrine ; the amine metabolites de- 
plete tissue stores of norepinephrine and/or com- 
pete with norepinephrine at receptor sites, thereby 
lowering blood pressure. Alpha-methyl-m-tyro- 
sine, a compound with similar biochemical proper- 
ties to Aldomet but which like Aldomet does not 
lower blood pressure in animals, has been found 
to have weak hypotensive properties in human 
hypertensives. 

We have continued to use monoamine oxidase 
inhibiting drugs, chiefly pargyline, in the manage- 
ment of hypertension in selected patients, either be- 
cause of resistance to other drugs or need for anti- 
depressive medication. Beneficial effects in 
patients with angina pectoris were previously 
ascribed to reduction of levels of blood pressure 
and pulse rate during exercise. It has now been 
shown in 7 patients that, as was presumed, the rise 
of cardiac output in response to standard exercise 
is reduced during pargyline therapy. 

An interesting area of study on vascular respon- 
siveness has appeared in studies of the pressor re- 
sponses to tyramine in patients with pheochromo- 
cytoma, and hypertensive subjects receiving Aldo- 
met or reserpine. A hypersensitivity to tyramine 
was found in three patients with pheochromocy- 



toma. This may be of sufficient magnitude to war- 
rant consideration of a tyramine-provocative test, 
analogous to the histamine test, but whose per- 
formance is not associated with any morbidity. 
Whereas, patients receiving reserpine exhibit di- 
minished tyramine sensitivity secondary to pre- 
sumed catecholamine depletion, several patients 
receiving Aldomet showed enhanced sensitivity. 
Our working hypothesis is that in patients with 
pheochromocytoma there may be increased levels 
of catecholamines, which are subject to tyramine 
release and an increased response. Similarly, a- 
methyl-amines, formed from Aldomet in tissues, 
could conceivably mediate enhanced tyramine 
pressor responses. 

Metabolism of Hydroxyproline and Collagen 

The urinary excretion of hydroxyproline 
(HOPr) peptides has been found to be a suitable 
index of the metabolism of soluble collagen in 
man, providing the subject ingests a gelatin-free 
diet. With dietary restriction, values in the range 
of 14—36 mg HOPr/day have been found in nor- 
mal adults. During the period of rapid growth 
(adolescence), values may be as high as 260 mg/ 
day. After growth ceases, there appears to be 
little change with increasing age ; this is contrary 
to our previous impressions from studies in which 
diet was not rigidly controlled. 

In addition to growth hormone, both thyroid 
hormone and parathyroid hormone have been 
found to increase HOPr excretion. This effect has 
been produced by administering the hormones to 
normal volunteer subjects and is also apparent in 
patients with thyrotoxicosis and hyperparathy- 
roidism. No other abnormalities have been en- 
countered in patients thus far ; HOPr excretion is 
normal in the so-called "collagen diseases". Pos- 
sibly, abnormalities will be detected in the latter 
states when the several HOPr peptides are iso- 
lated and quantified individually. 

Studies in growing mice have shown that lathy - 
ritic factor does not alter synthesis of total col- 
lagen but does produce an abnormally large pool 
of soluble collagen. Several anti-inflammatory 
drugs have shown no preferential effect on col- 
lagen versus total protein metabolism. Studies in 
several species have shown that arteries contain 
large amounts of collagen. This protein consti- 
tutes about 30 per cent of the total protein con- 
tent of aorta and up to 60 per cent of the protein 



NATIONAL HEART INSTITUTE 



107 



in muscular arteries. No change in ratio is seen 
during normal aging. 

Miscellaneous 

1. Studies are in progress to isolate and charac- 
terize the so-called angiotensinase(s) of blood. 
Angiotensin levels and disappearance rates are be- 
ing determined by a complex method which ter- 
minates in measurement of pressor activity in the 
rat. 

2. Gas-liquid phase chromatography has been 
used to detect minute quantities of the agly cones 
of digitoxin and digoxin. This, coupled with suit- 
able hydrolysis of the glycosides, may provide a 
means of accurate chemical assay of cardiac gly- 
cosides in biologic media. 

3. A large kindred has been studied in which 
primary pulmonary artery hypertension appears 
to be transmitted as a Mendelian dominant. 

4. Although the pressor response to norepineph- 
rine is increased in patients with hypertension, 
changes in plasma levels of free fatty acids fol- 
lowing injection of the amine are of the same mag- 
nitude as that in normal subjects. 

CARDIOLOGY BRANCH 

An important objective of this Branch is to 
identify the various mechanisms which regulate 
the heart's activity, and to assess their relative im- 
portance. In previous years the greatest emphasis 
was placed on defining the role of the ventricular 
end-diastolic pressure and volume in the determi- 
nation of the force of the heart's contraction. 
During 1962 greater attention was focused on the 
role of neurohumoral mechanisms in cardiovascu- 
lar regulation. It is now clearly recognized that 
the adrenergic nervous system is capable of pro- 
foundly altering the mechanical activity of the 
myocardium, and for this reason, considerable 
effort has been directed toward defining the oper- 
ation of this system. 

I. Studies on the Adrenergic Nervous System 

A. Biosynthesis and Metabolism of Norepineph- 
rine by the Heart 

Although the biosynthesis of norepinephrine has 
been demonstrated in vitro in tissue isolated from 
the adrenal medulla and from sympathetic nerve 
endings, it has recently been questioned whether 
the norepinephrine present in the heart is synthe- 



sized there or whether it is extracted from the 
blood by the sympathetic nerve endings in the 
heart. This problem was studied by perfusing an 
isolated dog heart with radioactive dopamine. 
Two to 12% of the added precursor was converted 
to norepinephrine in one hour. Thus, these studies 
show that the isolated heart is capable of synthe- 
sizing norepinephrine from dopamine. 

Although two enzymes (catechol-o-methyl trans- 
ferase and monoamine oxidase) have been shown 
to be capable of degrading norepinephrine, no 
evidence has been available regarding the rela- 
tive importance of these enzymes within the heart. 
This problem was investigated using an isolated 
dog heart in which the norepinephrine was labeled 
by the administration of tritiated norepinephrine. 
The o-methylate metabolite, normetanephrine, 
was demonstrated to be a major metabolic product 
in the heart; this finding emphasized the impor- 
tance of o-methylation in the metabolism of nor- 
epinephrine in the heart. 

B. Definition of the Subdivisions of ther Norepi- 
nephrine Pool 

Studies have also been continued to determine 
if the neurotransmitter is distributed homogene- 
ously or whether it is distributed into multiple 
functional compartments. Infusions of tyramine 
were given until tachyphylaxis to further adminis- 
tration of the drug was obtained. Cardioacceler- 
ator nerve stimulation was carried out before and 
after the tyramine infusion and no significant re- 
duction in the inotropic or chronotropic responses 
to stimulation occurred. Since nerve stimulation 
was evidently capable of releasing norepinephrine 
at a time when the store of norepinephrine avail- 
able to release by tyramine was depleted, it is clear 
that some of the "readily available norepineph- 
rine" can be released by nerve stimulation but not 
by tyramine. Thus, it is concluded that the 
"readily available norepinephrine" stores may be 
further subdivided functionally. 

Although radioactive norepinephrine can be 
extracted and stored at the sympathetic nerve 
ending, little information is available regarding 
its distribution within the neurotransmitter store. 
To investigate this problem radioactive norepi- 
nephrine was administered to dogs and the specific 
activity of norepinephrine was determined in 
coronary sinus blood before and during release of 
norepinephrine augmented by tyramine or car- 



108 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 






dioaccelerator nerve stimulation, and these values 
were compared with the values in myocardial tis- 
sue. It was found that upon augmented release 
of norepinephrine the specific activity of norep- 
inephrine in blood fell. However, in all experi- 
ments the specific activity of norepinephrine in 
the blood released from the heart exceeded the 
specific activity in the cardiac tissue. These obser- 
vations indicate that the radioactive norepi- 
nephrine is distributed unevenly within the 
endogenous store, initially mixing principally 
with the more exchangable norepinephrine. 

G. Pharmacology of Adrenergic Blocking Agents 

The function of the autonomic nervous system 
has also been studied with the aid of two anti- 
adrenergic drugs, reserpine and guanethidine. 
These agents are of interest since they are exten- 
sively utilized in clinical practice, yet their precise 
mechanisms of action have not been fully eluci- 
dated. It was concluded that the mechanisms of 
action of guanethidine and reserpine differ con- 
siderably. When guanethidine is administered 
the initial release of norepinephrine from the 
heart produces an adrenergic response, but this is 
associated with only minimal tissue depletion of 
norepinephrine. In contrast, resperpine produces 
more rapid depletion of myocardial norepineph- 
rine stores than does guanethidine, but it does 
not result in the release of norepinephrine from 
the heart into the coronary sinus blood. 

In other experiments the heart rate response to 
cardioaccelerator nerve stimulation and the cor- 
responding levels of myocardial norepinephrine 
content were determined and correlated at various 
time intervals following the intravenous injection 
of reserpine and of guanethidine. Guanethidine 
produced complete blockade of the cardiac accel- 
erator response before producing measurable de- 
pletion of myocardial norepinephrine content. In 
contrast, reserpine reduced the positive chrono- 
tropic response to cardio-accelerator nerve stimu- 
lation only after myocardial norepinephrine levels 
had been reduced to approximate 0.3 jug./gm. 
These observations suggest that the interference 
with adrenergic transmission produced by guan- 
ethidine is independent of changes in the level of 
stored adrenergic transmitter, but that almost 
complete depletion of stored adrenergic transmit- 
ter must occur before reserpine-induced adrener-i 
gic blockade develops. 



Although the cardiovascular response to those 
vasoactive amines which act by releasing endoge- 
nous norepinephrine is profoundly reduced by the 
chronic administration of reserpine, the response 
of these amines immediately after the administra- 
tion of reserpine has been poorly defined in the 
past. The cardiovascular responses to tyramine, 
tryptamine, metaraminol and amphetamine were 
found to be greatly potentiated for several hours 
after the intravenous administration of reserpine, 
while the responses to norepinephrine were essen- 
tially unchanged. The administration of reser- 
pine increased the quantity of norepinephrine 
released from the heart by tyramine. It was con- 
cluded that the potentiated norepinephrine release 
was not due to an increase in the size of the 
"tyramine releasable" compartment of norepi- 
nephrine, but that the reserpine facilitated the 
release of norepinephrine by tyramine by prevent- 
ing the reentry and/or binding of the norepi- 
nephrine. 

Although it is well recognized that reserpine 
can produce total depletion of norepinephrine in 
the cardiovascular system in the experimental 
animal and its anti-adrenergic action has been at- 
tributed to this effect, no information has been 
available regarding the effect of reserpine admin- 
istered clinically in man. In order to study this 
problem, biopsies of atrial appendages were ob- 
tained at the time of cardiac operation in 22 
patients who were in normal sinus rhythm and 
who had never been in congestive heart failure. 
The average norepinephrine concentration in these 
tissues was 1.87/xg./gm. In three patients who re- 
ceived 0.25-0.5 rag. reserpine orally for 30 days, 
the atrial norepinephrine concentrations were 
0.36, 0.10, and 0.04 /ig./gm. These results indicate 
that reserpine given orally to patients in the ordi- 
nary clinical doses significantly reduces the myo- 
cardial norepinephrine concentration. However, 
supramaximal electrical stimulation at the cardiac 
sympathetic nerves, at the time of operation, was 
still found to produce an adrenergic response. 

Previous studies showing in anesthetized open- 
chest dogs that guanethidine and reserpine block 
reflex venoconstriction were extended to man. 
The effects of three commonly employed antihy- 
pertensive agents, reserpine, guanethidine and 
alpha methyl DOPA, on reflex venoconstriction 
were studied. In all cases reflex venoconstriction 
was abolished after chronic administration of 



NATIONAL HEART INSTITUTE 



109 



these drugs in therapeutic doses. These effects 
which result in a decrease in venous return to the 
heart, appear to be important in the production 
of orthostatic hypotension in patients undergoing 
therapy with these drugs. 

D. The Autonomic Nervous System in Heart 
Failure, Shock and Anemia 

It has been shown previously that pharmaco- 
logic blockade of the adrenergic nervous system in 
normal human subjects interfered with the circu- 
latory response to muscular exercise. These stud- 
ies have now been extended ; an attempt was made 
to evaluate the contributions of adrenergic reflexes 
and of myocardial catecholamine stores to cardiac 
homeostasis in man by determining the effects of 
guanethidine in patients with borderline conges- 
tive heart failure. Guanethidine clearly increased 
the signs and symptoms of heart failure in five of 
10 patients, suggesting that the adrenergic nerv- 
ous system plays an important compensatory role 
in the circulatory adjustments of patients to con- 
gestive heart failure. These studies also empha- 
size the need for caution in the use of highly effec- 
tive antiadrenergic drugs in the treatment of 
patients with limited cardiac reserve. 

Although the sympathetic nervous system has 
been implicated as a major determinant of cardio- 
vascular function during muscular exercise in nor- 
mal man, its role in patients with congestive heart 
failure has not been defined. In an effort to asssay 
the activity of the sympathetic nervous system, 
arterial norepinephrine was measured in normal 
subjects and in cardiac patients both with and 
without congestive heart failure at rest and during 
exercise. These patients with congestive heart 
failure showed an augmentation of arterial nor- 
epinephrine which exceeded that of the normals in 
every instance. These results indicate that there 
may be an increased activity of the sympathetic 
nervous system during exercise in congestive heart 
failure and it was suggested that this activity may 
play an important supportive role to the 
circulation. 

An investigation was carried out in order to de- 
termine whether hemorrhagic shock, which is 
known to produce an increased activity of the 
sympathetic nervous system, alters the release of 
norepinephrine from the "readily available" nor- 
epinephrine store. The cardiovascular responses to 
norepinephrine infusion and to infusion of tyra- 



mine, which acts by releasing endogenous nore- 
pinephrine, were compared before hemorrhage 
and at various time intervals after hypotension. 
The pressor effects of tyramine were found to be 
markedly reduced after several hours of hypoten- 
sion. The responses to infusions of norepinephrine 
and angiotensin were only minimally attenuated 
during similar periods of hypotension. It there- 
fore appears that prolonged activation of the sym- 
pathetic nervous system by hypotension alters the 
release of norepinephrine from the "readily avail- 
able" store. 

In order to determine whether the autonomic 
nervous system is important in mediating the cir- 
culatory response to acutely induced severe ane- 
mia, the effect of this stimulus w T as compared in 
normal unanesthetized dogs, and in unanesthetized 
animals with denervated hearts. These studies in- 
dicate that the unanesthetized dog, when subjected 
to acute anemia, can increase its cardiac output 
despite the absence of a nerve supply to the heart. 
This elevation in output was achieved in the de- 
nervated animals primarily by an increased stroke 
volume, whereas in control dogs it was attributable 
primarily to increased heart rate. 

E. The Mechanism of Action of Sympathomimetic 
Drugs 

A detailed investigation on the mechanism of 
action of metaraminol, a widely used vasopressor 
agent in the clinical management of hypotension, 
was carried out in experimental animals and it was 
concluded that the positive inotropic and pressor 
responses to metaraminol are due primarily to the 
release of norepinephrine at adrenergic nerve 
endings. 

II. Studies on the Dynamics of Ventricular 
Contraction 

A. Gineradiographic Measurements of Ventricular 
Dimensions 

In order to analyze the mechanical activity of 
the left ventricle in man, a method for the precise, 
continuous measurement of ventricular dimen- 
sions in closed-chest subjects has long been sought. 
Silver-tantalum clips were sutured to the surface 
of cardiac chambers in patients undergoing car- 
diac surgery. After recovery cineradiograms were 
obtained and the distances between clips were 
measured on each individual frame of the film. 



110 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



There was an increase in right ventricular size 
with inspiration and a decrease with expiration. 
Changes in left ventricular dimensions were less 
marked and lagged 3-4 cardiac cycles behind the 
changes in the right ventricle. The administra- 
tion of isoproterenol and norepinephrine resulted 
in a decrease in the ventricular end-diastolic di- 
mensions while methoxamine produced an increase 
in end-diastolic dimensions. Exercise also pro- 
duced a consistent decrease in right and left ven- 
tricular dimensions, at a time when the rate of 
pressure development in the right ventricle was 
increased. This cineradiographic technique per- 
mits, for the first time, accurate and reproducible 
measurements of ventricular dimensions in man 
throughout the cardiac cycle and over the course 
of a number of cardiac cycles. 

B. Left Ventricular Function 

Although the response of the right heart to ex- 
ercise has been well documented, the study of left 
heart dynamics during exercise has not been pos- 
sible, until recently. The development of the 
transseptal method of left heart catheterization 
has permitted the introduction of flexible cathe- 
ters into the left heart by the venous route, and 
using this technique a study of the left heart re- 
sponses to standard exercise on a bicycle ergometer 
has been initiated. It appears that in patients 
with aortic stenosis a moderate increase in cardiac 
output is accompanied by little if any change in 
the pressure gradient across the aortic valve, b_v 
an elevation of left ventricular end-diastolic pres- 
sure, and by only a slight increase in left ventricu- 
lar systolic pressure, suggesting that in such 
patients the left ventricle has little "functional 
reserve." 

Ventricular function was also studied in patients 
by infusion of graded doses of angiotensin. If a 
response which is typical of the normal left ven- 
tricle could be established, the method could pro- 
vide a readily controlled stress mechanism for the 
evaluation of patients with compromised ventricu- 
lar function. In the patients studied so far, angio- 
tensin infusion has resulted in minimal changes 
in cardiac output, but sizeable increases in left 
ventricular stroke work and left ventricular end- 
diastolic pressure. It is anticipated that this 
method will have clinical value in the assessment 
of left ventricular function. 



III. Studies on Digitalis 

The mechanism of action of digitalis has been 
of considerable interest to investigators in this 
laboratory for several years. These studies were 
continued in two general directions : 

A, Effects of Digitalis on the Nonf ailing Human 
Heart 

Patients with acquired valvular heart disease 
and cardiac enlargement who were able to perform 
normal everyday activity without difficulty in the 
absence of digitalis therapy were exercised while 
receiving a placebo and again while receiving di- 
goxin. Varying degrees of exercise were per- 
formed on a treadmill in a metabolic chamber and 
oxygen consumption was measured continuously. 
In all patients the oxygen debt was smaller dur- 
ing the period of digoxin administration although 
the external work performed was identical, indi- 
cating that the functional status of their circu- 
latory system was improved by the drug. On the 
basis of this study it appears that digitalis ad- 
ministration is beneficial to at least some patients 
who have cardiac disease and enlarged hearts and 
some decrease in cardiac reserve without signs or 
symptoms of heart failure. 

Since digitalis glycosides fail to elevate the 
cardiac output in patients who are not in heart 
failure, it has been suggested that these drugs do 
not stimulate myocardial contractility in nonfail- 
ing hearts. Accordingly, an investigation was 
carried out in order to determine whether ouabain, 
administered intravenously, modifies the force of 
contraction of normal, or near normal, nonf ailing 
hearts. Changes in myocardial contractility were 
assessed by continuously recording the rate of 
change of intraventricular pressure (dp/dt). In 
10 patients without heart disease peak dp/clt in- 
creased by 10 to 49 percent of control values. In 
the absence of changes in intraventricular systolic 
and diastolic pressures and in heart rate, the ob- 
served increases in dp/dt may be assumed to re- 
flect increases in myocardial contractility. These 
observations in intact, unanesthetized subjects 
therefore indicate that ouabain is capable of stim- 
ulating the contractility of the nonfailing and the 
normal human heart. 

The effects of digitalis on the peripheral vascu 
lar system of man were investigated. In normal 






NATIONAL HEART INSTITUTE 



111 



subjects ouabain resulted in an increase in both 
arterial and venous tone, while in patients with 
overt cai'diac failure there was a decrease in both 
arterial and venous tone. These observations help 
to explain a number of the actions of digitalis 
which have puzzled physicians for many years. 

B. Determinants of the Effectiveness of Cardiac 
Glycosides 

Others have suggested that the positive ino- 
tropic effect of ouabain is dependent upon an in- 
tact myocardial catecholamine store and that the 
amount of ouabain required to produce fatal ar- 
rythmias is influenced by myocardial norepine- 
phrine content; the exact mechanism by which 
glycosides increase the refractory period of the 
atrioventricular conduction system has been un- 
known. We have shown that intravenous ouabain 
infusion produced comparable increments in myo- 
cardial contractile force in control dogs, in dogs 
with chronic cardiac denervation, in dogs with 
myocardial catecholamine depletion, in vagoto- 
mized, reserpinized dogs, and in dogs with acute 
section of preganglionic sympathetic pathways. 
It was also determined that there were no sig- 
nificant differences in the toxic doses of ouabain 
in each of these four groups of animals. In addi- 
tion, it has been shown that ouabain infusions pro- 
duce the greatest prolongation of the A-V func- 
tional refractory period in control dogs, and that 
this effect was reduced in vagotomized dogs and 
reduced further in the dogs with chronic cardiac 
denervation. These observations indicate that the 
positive inotropic and arrhythmic doses of oua- 
bain are independent of myocardial catechloamine 
stores, but that a large portion of the prolongation 
of the A-V functional refractory period pro- 
duced by ouabain is dependent upon intact cardiac 
adrenergic innervation. 

The difficulties encountered in the management 
of thyrotoxic heart disease with digitalis have 
suggested that changes in metabolic activity in- 
duced by the thyroid state may condition the myo- 
cardial response to digitalis. It was observed that 
the increase in ventricular contractile force pro- 
duced by ouabain was not significantly different 
in euthyroid and hyperthyroid animals. However, 
ouabain produced a significantly greater increment 
in the ventricular contractile force of hypothyroid 
animals. The toxic doses of ouabain were identical 
in euthyroid and hypothyroid dogs, but were sub- 



stantially greater in hyperthyroid dogs. In addi- 
tion, hyperthyroid dogs were more resistant than 
euthyroid or hypothyroid dogs to the prolongation 
of the refractory period of the A-V node. These 
observations indicate that the three myocardial 
effects of ouabain studied were not altered in a 
uniform manner by changes in the thyroid state. 
In addition they help to explain some of the prob- 
lems encountered by the physician in the manage- 
ment of the thyrocardiac patient. 

IV. Cardiovascular Physiologic Studies 

As before, the cardiovascular physiologic inves- 
tigations have centered on the coronary circulation. 
The effect of varying coronary blood flow on myo- 
cardial oxygen consumption was studied in 23 
experiments. The ventricles were kept empty, 
developed no pressure and performed to external 
work, while their temperature was held constant. 
A comparison of myocardial oxygen consumption 
at two levels of coronary blood flow (and oxygen 
delivery) was made in 42 instances, and in 32 of 
them myocardial oxygen consumption increased 
substantially as coronary blood flow was elevated. 
The ten exceptions all occurred when oxygen de- 
livery greatly exceeded myocardial oxygen con- 
sumption, with oxygen extraction ratio below 
35%. The energy derived from anaerobic metabo- 
lism at low levels of coronary blood flow could not 
account for the lower values of myocardial oxygen 
consumption which occurred under these circum- 
stances. These observations show that in a non- 
working heart, when myocardial oxygen extraction 
is in a physiologic range, myocardial oxygen con- 
sumption is intimately dependent on coronary 
blood flow and suggest that this dependence should 
be considered in the interpretation of experiments 
on the determinants of myocardial oxygen con- 
sumption. In seven experiments performed on 
hearts which developed pressure during systole, 
myocardial oxygen consumption remained constant 
in spite of large changes in coronary blood flow. 
Further experiments are planned to define the 
mechanisms responsible for the differences ob- 
served in the empty heart and the heart which is 
forced to develop a systolic pressure. 

Efforts have also been directed toward the 
development of methods for the continuous 
measurement of local myocardial blood flow. 
Progress has been made in the construction of a 
heated thermistor probe, with which changes in 



112 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



perfusion modify the rate of heat transfer from 
the probe. A series of experiments have been ini- 
tiated together with investigators in the Labora- 
tory of Kidney and Electrolyte Metabolism, NHI. 
Gaseous hydrogen, dissolved in saline, is injected 
into the coronary circulation, and the rate at which 
the hydrogen is removed from the heart deter- 
mined by means of platinum electrodes introduced 
into the myocardium as well as into the coronary 
venous blood. Preliminary results indicate that 
both the thermistor and hydrogen techniques show 
considerable promise, and it is hoped to perfect 
these methods so that they can be employed for 
the measurement of myocardial blood flow in man. 
Nonartifactual pressure tracings were obtained 
at catheterization in intact, unanesthetized human 
subjects. These tracings were analyzed for their 
first 50 harmonic components by the Computation 
and Data Processing Branch. It appears that the 
major harmonics of such tracings are within the 
first five to six calculated harmonics, and these 
harmonics are included within the frequency re- 
sponse (8 cycles/sec.) of standard catheter- 
external manometer systems. 

V. Studies in Clinical Cardiology 

A number of clinical investigations on patients 
with congenital and rheumatic heart disease were 
completed in 1962. As before, many of these 
studies were carried out in conjunction with the 
Clinic of Surgery, NHI. During the past year, 
attention was focused on patients with left ven- 
tricular hypertrophy, with and without obstruc- 
tion to left ventricular outflow. The clinical fea- 
tures, phonocardiographic, roentgenologic and 
electrocardiographic findings in 100 patients with 
congenital aortic stenosis were correlated with the 
severity of obstruction as determined by left heart 
catheterization. In addition to defining the spe- 
cific value of each clinical finding in the assessment 
of the severity of obstruction, indications for sur- 
gical correction of this lesion were established and 
the clinical and hemodynamic effects of operation 
were analyzed. 

Although the definitive determination of the 
site and severity of obstruction to left ventricular 
outflow must be left to catheterization techniques, 
a simple and innocuous test would be of consider- 
able value in the evaluation of such patients. The 
first derivative of the pressure pulse was continu- 
ously calculated by an electronic differentiating 



analogue computer. Patients with hypertrophic 
subaortic stenosis were found to have a brachial 
artery peak dp/dt greater than that found in nor- 
mal objects. This appears to be due to the fact 
that little or no obstruction exists in early systole. 
In contrast, patients with fixed obstruction to left 
ventricular outflow (valvular and discrete mem- 
branous subvalvular stenosis) exhibited obstruc- 
tion to left ventricular outflow throughout all of 
ventricular systole and the brachial artery peak 
dp/dt proved to be lower than normal. It is an- 
ticipated that the technique described should 
prove to be a simple and practical one in the recog- 
nition and differentiation of the various forms of 
obstruction to ventricular outflow. 

It has recently been found that a unique feature 
of the disease in patients with idiopathic hyper- 
trophic subaortic stenosis is the inconstant nature 
of the obstruction. Prominent among the deter- 
minants of the severity of obstruction is the con- 
tractile state of the myocardium. It therefore 
seemed likely that the administration of sympath- 
omimetic drugs could exert profound effects on 
the circulatory dynamics of patients with hyper- 
trophic subaortic stenosis. The intravenous ad- 
ministration of isoproterenol generally lowered 
the cardiac output and the systemic arterial pres- 
sure, while elevating the left ventricular systolic 
and end-diastolic pressures. Thus, this drug con- 
sistently decreased the effective orifice within the 
left ventricular outflow tract. In contrast, meth- 
oxamine abolished the left ventriculo-arterial pres- 
sure gradient in four patients. 

The clinical, hemodynamic, and angiocardiogra- 
phic findings in a group of 11 patients with idio- 
pathic myocardial hypertrophy were described. 
These patients were either asymptomatic or their 
symptoms showed little if any progression over 
many years. The clinical findings and evidence of 
left ventricular hypertrophy on electrocardiogra- 
phic, roentgenologic and angiocardiographic ex- 
aminations were similar to the findings in patients 
with idiopathic hypertrophic subaortic stenosis. 
In contrast, none of these patients exhibited any 
hemodynamic evidence of obstruction to blood 
flow during the control state. The close relation- 
ship between this clinical syndrome and hypertro- 
phic subaortic stenosis was shown by the finding 
that a gradient between the left ventricle and a 
systemic artery could be provoked by the adminis- 
tration of isoproterenol in these patients. 



NATIONAL HEART INSTITUTE 



113 



It has been generally thought that significant 
elevations of the left atrial and plumonary vascu- 
lar pressures occur in patients with mitral regur- 
gitation of sufficient severity to produce serious 
disability and gross enlargement of the left atrium. 
A group of patients with severe mitral regurgita- 
tion in whom gross left atrial enlargement was 
accompanied by normal left atrial and pulmonary 
artery pressures was described. The observed dis- 
crepancy between left atrial size and pressure must 
reflect a disturbance in the compliance of the left 
atrial wall. It was suggested that long-standing 
mitral regurgitation modifies the mechanical char- 
acteristics of the atrial wall and that the presence 
of a normal left atrial pressure must not be as- 
sumed to exclude the presence of severe mitral re- 
gurgitation. 

Other Remarks 

A substantial fraction (approximately 25%) of 
the professional and technical efforts of the Cardi- 
ology Branch are devoted to clinical activities 
which are not directly related to the direct aims 
of its research program. These nonresearch ac- 
tivities include: (1) Recording, mounting and in- 
terpretation of all of the electrocardiograms for 
the Clinical Center. Approximately 6,000 trac- 
ings were handled in 1962. In addition, a course 
in electrocardiographic interpretation was given 
and personal instruction in ECG interpretation 
was provided to Clinical Associates from other 
laboratories of NHI and from other institutes. (2) 
Clinical cardiology consultations for the Clinical 
Center. (3) Cardiology consultations to the Clinic 
of Surgery, NHI. (4) An average of 3 post-opera- 
tive cardiac catheterizations weekly, carried out 
for the Clinic of Surgery. (5) Consultations in 
pulmonary physiology, and performance of pul- 
monary function tests for the Clinical Center. 

Section on Clinical Biophysics 

A. Cardiovascular Activities 

The immediate objectives of the Section on 
Clinical Biophysics have been to learn as much 
as possible about the variables controlling the me- 
chanical behavior of the various components of 
the cardiovascular system. The ultimate objective 
is synthesis of this information into an integrated 
picture of overall cardiovascular system behavior 
with emphasis on the interaction of its components. 



This implies the necessity of clearly identifying 
unique sets of controlling variables experimentally 
so that appropriate systems of mathematical equa- 
tions may be developed. 

The experimental studies of these systems of 
variables may be categorized in the following 5 
groups: (1) Heart, (2) Great Vessels, (3) Small 
Vessels, (4) Veins, and (5) Control Systems. Al- 
though certain exploratory studies under (4) and 
(5) have been carried out, the major activities 
over the past year have been concentrated in the 
first 3 categories : 

(1) Heart: Studies designed for the prelimi- 
nary identification of variables that uniquely de- 
scribe myocardial mechanical behavior have been 
interesting and rewarding. The general approach 
to this problem has been to hold all parameters 
(e.g., temperature, metabolic milieu, etc.) constant 
measure all variables (e.g., ventricular pressure, 
ejection rate, etc.), and then analyze the relation- 
ship between any two variables for various selected 
constant values of the remaining variables. If 
unique relationships emerge between variables 
taken in this way, (two at a time) one has then 
identified primary variables describing the sys- 
tem. Specifically, the following studies have been 
done. 

An animal preparation was devised in which it 
was possible to study the myocardial mechanical 
behavior in an intact, metabolically controlled, 
normally beating heart by the use of a heart-lung 
machine. Instrumentation was developed which 
made possible the computation of instantaneous 
tension and shortening velocity or rate of strain 
of the muscle elements contained in a selected 
circumference of the myocardium at different in- 
stantaneous heart volumes or fiber lengths. 

Using this methodolgy, the tension-velocity data 
obtained were analyzed by computing regression 
equations relating wall tension to rate of strain 
at selected constant instantaneous heart volumes. 
A reciprocal relationship between the wall tension 
and rate of shortening of the wall emerged from 
this analysis. It therefore appears that 3 primary 
variables describing myocardial mechanical be- 
havior are (1) instantaneous fiber length, (2) in- 
stantaneous rate of shortening and (3) instantane- 
ous wall tension. It is interesting to note that 
during these studies many different stroke works 
would occur from identical initial fiber lengths. 
These findings are not consistent with the concept 



114 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



that Starling's law uniquely describes myocardial 
behavior. These findings are consistent with the 
concept that myocardial function is determined 
by intrinsic muscle laws perhaps similar to those 
described by A. V. Hill for skeletal muscle. 

There was sufficient variance in the foregoing 
data to make premature any statement that all of 
the primary variables had been identified. In 
view of the indirect approach that was necessary 
to measure these variables, it was highly desirable 
to confirm the above findings by direct measure- 
ment of wall tension, shortening velocity and fiber 
length in the normal beating heart. Transducers 
for this did not exist and therefore a program of 
instrument development was instituted. Trans- 
ducers have just been developed in this section 
which can measure elongating strains on the sur- 
face of the heart as well as elongating strains 
through the heart wall (that is the increase in 
wall thickness as a function of time). Trans- 
ducers were also developed to measure shearing 
strains in the wall, that is, the angular displace- 
ment of one muscle layer with respect to the other. 
Finally, a force or tension measuring transducer 
was devised. Although physiological studies us- 
ing these transducers are just now getting under 
way, preliminary results indicate that the heart 
muscle undergoes relatively small shearing strains 
during the ejection phase of systole. The degree 
of shear is somewhat greater in the axis from apex 
to base than circumferentially about the lesser 
radius. The usual magnitude is of the order of 
1 degree maximum shear over the ejection period. 
This is considered small. The change in wall 
thickness during ejection phase is of the order of 
5%. Circumferential and longitudinal elongating 
strains are of the same order of magnitude. These 
studies should culminate in a body of knowledge 
Avhich will allow precise statements regarding the 
relationships among the primary variables con- 
trolling the mechanical behavior of the myocar- 
dium. Such statements open the way to mathe- 
matical generalizations regarding myocardial 
function. 

(2) Great Vessels: From a purely physiologi- 
cal point of view the information obtained in the 
foregoing studies indicate that the response of 
the heart from a given end-diastolic fiber length 
depends on the time course of the load presented 
to the ventricles. This raises the question as to 
the nature of the load presented to the myocar- 



dium by the great vessels. Since the vascular 
system contains inertial, viscous, and elastic com- 
ponents the load presented by the great vessels 
to the heart will contain components of force re- 
lated to these properties. Loads of this nature 
cannot be defined by the simple concept of periph- 
eral vascular resistance, but must be defined with 
the use of hydraulic impedance functions. Hy- 
draulic impedance functions relate the instanta- 
neous value of pressure to flow. This is done by 
resolving the respective pressure and flow curve 
into its Fourier series and then calculating the 
complex ratio between each pressure harmonic and 
its corresponding flow harmonic. The impedance 
patterns in the pulmonary artery and the aorta 
appear qualitatively similar. The impedance 
magnitudes initially diminished rapidly with fre- 
quency to wax and wane in magnitude thereafter. 
Such patterns are consistent with the view that 
both systems are distributed wave transmitting 
systems. The waxing and waning of amplitudes 
is consistent with significant reflections of energy 
from the periphery and other vessel junctions. 

Carrying these studies one step further, the in- 
stantaneous relationship between the flow and the 
pressure-gradient was determined. In this case 
the calculation of the impedance series relating 
the pressure gradient to flow yields the "distrib- 
uted longitudinal impedance" of the system, a 
property independent of a wave reflection and de- 
pending only on the local properties of the blood 
and vessel. It follows that knowledge of these 
relationships should permit calculation of insta- 
taneous blood flow from more simply obtained 
pressure-gradient information which would be 
of obvious value in clinical cardiovascular dy- 
namic studies. The distributed impedance of the 
major vessels was analyzed not only with respect 
to the behavior of the impedance magnitudes with 
frequency but also with respect to the behavior of 
the real and imaginary parts of each term. A de- 
tailed analysis of these data indicate a striking 
agreement with previous theoretical considera- 
tions, a finding representing 'a major advance in 
the field of circulatory dynamics. 

To supplement the foregoing studies detailed 
measurements of the instantaneous relationship be- 
tween blood vessel diameter and instantaneous 
lateral pressure have been carried out again using 
the mathematical techniques outlined above. The 
impedance series so calculated will represent the 






NATIONAL HEART INSTITUTE 



115 



"distributed shunt impedance" of an energy trans- 
mitting system. The frequency spectrum of these 
impedance f unctions indicates that the blood vessel 
wall acts as a viscoelastic body containing inertia! 
components also. Similar patterns have been found 
from the ascending aorta down to the lower ab- 
dominal aorta. Certain mathematical relation- 
ships exist between this "shunt impedance" and the 
"longitudinal impedance" of a blood vessel (dis- 
cussed above) which permit one to relate precisely 
the pressure gradient, the flow, the pressure, and 
the pulse wave velocity to the dimensions and 
physical properties of the vascular bed. Studies 
to permit us to take this final step in establishing 
the mechanical behavior of the great vessels are 
under way, however, without results as yet. 

(3) Small Vessels : Study of the fluid dynamics 
of the small systemic blood vessels has been only 
exploratory in nature and has been confined pri- 
marily to the "waterfall effect" in the lung. The 
"waterfall" effect in the lung refers to the finding 
that blood flow through the lung for a given pul- 
monary artery pressure will be independent of the 
left auricular pressure until the left auricular pres- 
sure equals the airway pressure. A mathematical 
theory based on certain assumptions has been 
evolved to explain this phenomenon. Studies are 
at present being done in a physical model to estab- 
lish the validity of the theory in the model and to 
define critical parameters that will be necessary to 
be measured in the living pulmonary circulatory 
system. The results to date are in the process of 
analysis ; however, qualitatively it has been demon- 
strated that the pressure-flow relationship in the 
model behaves like that in the pulmonary circula- 
tory system. 

B. Studies on Puhnonary Mechanics 

It has been shown previously from this section 
that a functional relationship exists between the 
transpulmonary pressure, respiratory gas flow, and 
the degree of lung inflation. The relationship be- 
tween the maximum expiratory flow and the degree 
of lung inflation (or volume) is of special interest 
and has been termed the maximum expiratory flow 
volume curve (F-V curve). Over the upper part 
of the vital capacity the relationship between the 
maximum expiratory flow and the degree of in- 
flation is effort dependent and influenced primarily 
by the resistance of the upper airways. Over the 
lower segment of the curve, which has been termed 
the a F-V curve, the flow is not dependent on 

705—685 — 63 9 



either maximum expiratory effort or on upper air- 
way resistance. Theoretically this part of the 
curve depends upon the resting dimensions and 
physical properties of the lung and intrathoracic 
airways as well as the physical properties of the 
alveolar gas. Any acceptable mathematical model 
of the lung must be able to predict the changes in 
the a F-V curve that would be brought about by 
specified changes in the physical properties of the 
alveolar gas. Therefore, studies were designed in 
which the physical properties of the alveolar ex- 
pired gas could be measured. Various different 
mixtures of gas were given so that wide extremes 
of both viscosity and density were achieved. It 
was found that the maximum flow that can be 
achieved over the lower part of the vital capacity 
is significantly more dependent on gas viscosity 
than on density. The converse was true for the 
peak expiratory flow which can be achieved over 
the upper part of the vital capacity. Thus, peak 
flow is relatively independent of viscosity but de- 
pends heavily on density. Certain problems re- 
lated to computer technology have arisen that have 
prevented further progress on the detailed "fit- 
ting" of the a F-V curve to various mathematical 
models. Nevertheless, it has been possible to study 
certain limiting conditions predicted by some of 
these mathematical models. One of the models 
predicts that the maximum slope of the a F-V 
curve will vary inversely with the cube root of the 
density of the gas breathed and be relatively inde- 
pendent of the gas viscosity. "Blind" studies were 
done in which various observers were requested to 
measure the maximum slope on the a F-V curves. 
The measurements from each curve were then aver- 
aged. The average slope measurement from each 
of these curves was found to vary closely with the 
reciprocal of the cube root of the density. If a 
valid mathematical model can be developed, the 
way is open for applying computer analysis to 
mass surveys using these simply obtained a F-V 
curves. The potential importance of this to public 
health and air pollution matters is obvious. 

CLINIC OF SURGERY 

The investigative projects of the Surgery 
Branch have, as in past years, centered largely 
around development of new or improved methods 
for the surgical treatment of patients with con- 
genital or acquired heart disease. In general, 
these projects which have been carried out at both 



116 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the experimental and clinical levels, have been 
designed to elucidate the physiologic changes 
which occur as the result of various malforma- 
tions and the alterations which accompany com- 
plete (or incomplete) surgical correction. There- 
suits of appropriate work in the experimental 
laboratory are applied in the clinical program 
where opportunity is taken to make physiologic 
observations before, in the course of, and follow- 
ing cardiac operations. 

Within the past year, an increasingly large pro- 
portion of patients operated upon have been those 
with acquired rather than congenital heart disease. 
This difference in patient material probably re- 
flects the development of satisfactory operations 
for the correction of acquired stenotic and regurgi- 
tant lesions of the mitral and aortic valves. A 
recent analysis was made of the operative results 
in 50 patients with acquired calcific aortic stenosis 
who were subjected to detailed hemodynamic 
studies both before and after operation. The re- 
sults of the study indicated that debridement of 
the calcific valve followed by commissurotomy is 
the surgical procedure of choice in those patients 
in whom it is applicable. On the other hand, par- 
tial or total replacement of the aortic valve was 
found to be necessary in a significant proportion 
of all patients with this disease. 

Total prosthetic replacement of the aortic valve 
has been carried out in nearly 50 patients with con- 
genital or acquired valve disease. A tricuspid 
valve made of Teflon has been most often em- 
ployed and during short periods of followup has 
been shown to afford complete relief of outflow ob- 
struction and to be entirely competent. At present 
valves constructed of plain Teflon fabric and 
fabric which has been coated with a Teflon disper- 
sion are being used in patients to determine if 
coating will delay or prevent stiffening of the 
leaflets. 

In patients with predominant aortic stenosis be- 
fore operation we have observed large pressure 
gradients between the left ventricle and aorta in 
the early postoperative period. It is unlikely 
that stiffening of the prosthetic valve can occur 
within a few weeks and it seems likely that pa- 
tients demonstrating this phenomenon may have 
hypertrophic subaortic stenosis secondary to mas- 
sive hypertrophy of the left ventricle caused by 
the valvular lesion. A physiologic assessment of 
this lesion may provide us with further informa- 



tion about the hypertrophic form of subaortic 
stenosis which is, of course, also seen as an isolated 
lesion. The operative treatment of the primary 
form of hypertrophic stenosis has been improved 
within the last year and in the last three patients 
operated upon large masses of the enlarged muscle 
have been removed by means of a new and spe- 
cially designed instrument and immediate correc- 
tion of the hemodynamic abnormality has been 
achieved. This is in contrast to the earlier pa- 
tients in whom a less radical procedure was car- 
ried out and hemodynamic improvement appeared 
to occur gradually. 

In virtually all patients with calcific aortic 
stenosis the valve is immobile and the fixed orifice 
of the valve, in addition to being stenotic, permits 
a greater or lesser degree of aortic regurgitation. 
This clinical observation gave rise to speculation 
concerning the magnitude of regurgitation 
through various fixed orifices and suggested an 
experimental study in which regurgitant flow 
through the aortic valve was directly measured. 
Regurgitation was acutely induced in dogs by 
means of special cannula© which permitted a fixed 
regurgitant orifice to be created and closed at will. 
Retrograde flow, as well as the various components 
of forward flow, were measured with an electro- 
magnetic flowmeter in the aorta and femoral ar- 
tery. It was found that the area of the regurgitant 
orifice bore an almost linear relationship to the 
volume of regurgitant flow and that the changes 
in the pattern of flow in the aorta were accurately 
reflected in the flow pattern recorded in the femo- 
ral artery. The observations have been extended 
in 25 patients in whom the femoral flow pattern 
was determined directly at the time of operation. 
In patients without aortic valve disease there was 
no retrograde flow in the femoral artery at any 
time. In all patients with clinical evidence of 
aortic regurgitation, however, retrograde flow 
could be measured during diastole and its magni- 
tude correlated well with the area of the orifice 
through the incompetent valve which was meas- 
ured at subsequent operation. 

A clinical and hemodynamic analysis was made 
of the results of closed mitral commissurotomy in 
an unselected group of 35 patients subjected to 
operation. The status of the valve was assessed 
by left heart catheterization before and one year 
after operation. The results of the study indi- 
cated that the principal factor which determined 



NATIONAL HEART INSTITUTE 



117 



the extent to which closed mitral commissurotomy 
restored normal hemodynamics was the anatomic 
status of the valve encountered at operation. 
When a flexible, mobile and noncalcified valve was 
present, virtually all patients received dramatic 
hemodynamic benefit and quite often entirely nor- 
mal valve function could be restored. When, how- 
ever, immobility of the posterior leaflet or both 
leaflets was present closed operations afforded lit- 
tle or no benefit in the majority of the patients. 
The various clinical factors indicating preopera- 
tively that such an unfavorable valve might be 
present were also determined and the principal 
one was radiographic evidence of calcification of 
the valve. These findings have strengthened our 
impression that all predominantly regurgitant 
lesions of the mitral valve must be treated by valve 
replacement and, at this time, it is also felt that 
the calcified stenotic valve can be corrected only 
by this means. Within the past year 15 patients 
with regurgitant or stenotic lesions of the mitral 
valve have been operated upon and the mitral 
valve replaced with the ball prosthesis devised by 
Starr. Thirteen of the fifteen patients have sur- 
vived operation and all have shown striking clini- 
cal benefit. A number of them have also been sub- 
jected to detailed postoperative hemodynamic 
assessment and, although they are found to have 
normal left atrial pressure at rest, the prosthetic 
valve which has been employed has been shown to 
be stenotic. The small gradient evident across the 
prosthetic Starr valve may be responsible for the 
occurrence of embolization in patients who are not 
given anticoagulants and the hemodynamic data 
will give impetus to a modification of the design 
of the valve which will permit it to have a larger 
effective orifice. 

A significant proportion of the work in the 
experimental laboratory has centered around the 
problems of materials for prosthetic heart valves. 
Evidence has been collected, from the use of pros- 
thetic materials in vascular grafts, that the size 
of the pores in the prosthetic material may deter- 
mine the degree of fibrous tissue ingrowth and 
consequently the stiffening of such materials. 
Since stiffening must be avoided in prosthetic 
valves, the relationship of pore size to tissue 
ingrowth is under investigation in dogs. A new 
fabric composed of alternating fibers of collagen 
and dacron is used to replace part of the heart wall 
or a portion of a mitral or tricuspid valve leaflet. 



This fabric, when implanted, has a small pore size 
but as the collagen is absorbed a large pore size 
results. Applications of such combined natural 
and prosthetic materials for the use in heart valves 
will undoubtedly be forthcoming. 

Any material used for a prosthetic valve is sub- 
jected to stress, and fracture of leaflets due to 
flexing is a problem which has been encountered 
clinically. A plastic known as polypropylene is 
known to have an extremely long flex life but little 
information has been available concerning its bio- 
logic characteristics or its suitability for the use 
in heart valves. This material is being woven into 
suitable fabrics which are being tested as to their 
promotion of blood clotting, foreign body reaction, 
and possible toxic manifestations. The surface of 
a prosthetic material used for heart valves is also 
of importance and this is being investigated by 
the implantation, in animals, of dacron sheets 
which have been coated with other polymers so 
that some of them have slick (closed cell) surfaces 
and others porous (open cell) surfaces. The mate- 
rials are being tested by implanting triangular 
pieces of them in the outflow tract of the right 
ventricle and, in addition, a simulated valve leaflet 
is implanted so that it lies within the ventricle. 
The experiments should give information concern- 
ing not only the desirable surface characteristics 
of the fabrics but also the behavior of prosthetic 
leaflets made of each type. 

At the present time, the implantation of an arti- 
ficial valve, either mitral, aortic, or tricuspid in 
a patient is a long operative procedure and much 
of the time is necessitated by the placing and tying 
of sutures to anchor the valve. In previous re- 
ports a plastic adhesive has been described which 
has been found useful as a hemostatic agent. Work 
is continuing with this material concerning its 
reactions when implanted in tissue and, in collabo- 
ration with a plastics chemist, attempts are being 
made to improve the chemical formulation of the 
adhesive so that it, or a similar compound, may 
be used to cement artificial heart valves in place 
and/or to close intracardiac openings without the 
use of suture material. Within the heart this 
technique would, of course, obviate many of the 
present clangers in regard to complete heart-block 
caused by placement of sutures near the bundle of 
His. 

The vast majority of operations carried out for 
the correction of heart disease now require the use 






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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of extracorporeal circulation and several clinical 
and experimental projects have centered around 
this technique. In previously described studies it 
was found that animals subjected to extracorporeal 
circulation showed a significant loss of digitalis 
from the myocardium after 30 minutes of bypass. 
During the past year these observations were ex- 
tended to the study of patients and similar con- 
clusions were reached. Patients were given tritium 
labeled digoxin before operation and the radio- 
activity of myocardial biopsies was determined 
before and after bypass. An average decrease of 
myocardial radioactivity of 15% was observed 
while the radioactivity of the blood of the patient 
and heart-lung machine increased more than four 
times after the procedure. The loss of digoxin 
from the heart was not related to the duration of 
the bypass and it seems likely that the loss is 
accounted for by an acceleration in the normal 
metabolism of the compound. 

At the National Heart Institute, as in most cen- 
ters, homologous blood is used as the priming sub- 
stance in the artificial heart and lung machine. 
Several undesirable sequelae have been noted after 
bypass such as fever electrolyte' derangement, hy- 
potension, etc. Work in other laboratories has in- 
dicated that these changes may be due to the 
"homologous blood syndrome." This is being eval- 
uated experimetnally and an attempt is being 
made to isolate the component in homologous dog 
blood which is responsible for the shock-like pic- 
ture which often accompanies animal perfusion. 
The age of donor blood, its electrolyte content, and 
the relative effects of plasma and red cells in pro- 
ducing the syndrome are being studied. Prelimi- 
nary results indicate that the "toxic" factor is 
probably in the serum and that when washed red 
cells are used in the priming solution many of the 
undesirable accompaniments of bypass can be 
avoided. 

Many patients with intracardiac communica- 
tions have secondary pulmonary hypertension and, 
in the past, many of these patients were excluded 
as operative candidates because of the high risk 
attendant upon closure of the defect in this situa- 
tion. In such patients the circulatory shunt is 
usually a bidirectional one and in periods of stress 
or heart failure the right heart is able to decom- 
press itself through the intracardiac communica- 
tion. Abrupt closure of the defect may abolish 
this safety valve mechanism and lead to acute heart 



failure in the postoperative period. An extensive 
experimental study has been carried out concern- 
ing the use of perforated patches of prosthetic 
material which will allow gradual closure of intra- 
cardiac communications. In dogs it was found 
that perforations of less than 7 mm. in diameter 
in Ivalon closed within 10 days, while closure of 
8 mm. openings did not occur for many weeks. 
This technique has been applied in four patients 
with atrial septal defects, extreme pulmonary hy- 
pertension, and bidirectional shunts. In each of 
them perforated prostheses were used to close the 
defect and serial hemodynamic studies were car- 
ried out postoperatively. It was found that 6 mm. 
perforations closed within 10 days but 8 mm. per- 
forations remained open. All the patients sur- 
vived and it is felt that the decompression allowed 
by the perforated prosthesis was the determining 
factor in the success of the procedures. 

Clinical assessment was also made of the changes 
in the pulmonary artery pressure and pulmonary 
vascular resistance in 29 patients who had pul- 
monary hypertension secondary to intra-or extra- 
cardiac communication. All of the patients were 
studied by means of cardiac catherization before 
and at intervals after operation. All had mean 
pulmonary artery pressure greater than 50 mm. 
Hg preoperatively. The pulmonary artery pes- 
sue fell significantly in all patients with extracar- 
diac shunts and the pulmonary resistance was 
normal in all but one of these patients. In 17 pa- 
tients with intracardiac shunts, however, the pul- 
monary artery pressure fell far less and elevation 
of both pulmonary artery pressure and pulmonary 
vascular resistance persisted postoperatively in 
virtually all of them. 

Complete heart-block is an unfortunate compli- 
cation of the operative treatment of various intra- 
cardiac malformations, particularly ventricular 
septal defect and the various forms of persistent 
A-V canal. Although appropriate electrical de- 
vices for maintaining the ventricular rate in such 
patients are now available, the chief limitation to 
their use has been electrode breakage. In cooper- 
ation with the Instrument Fabrication Section a 
device has been developed for testing various types 
of wire electrodes by flexing them extremely rap- 
idly and for long periods of time. Initial obser- 
vations indicate that a greatly improved myocar- 
dial electrode may be constructed from Elgiloy, an 
alloy used commercially for watch springs. Other 



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studies on complete heart-block and its physio- 
logic manifestations have concerned the reaction 
of animals to shock when their heart rate has been 
controlled. In a normal animal, when shock has 
been induced by bleeding, the heart rate rises strik- 
ingly and after the shock-state has been main- 
tained for some period of time, the reinfusion of 
blood is ineffective. It has been found that if the 
heart rate of the dog is controlled during the 
shock-state, by means of induced block and an 
electrical pacemaker, the recovery from prolonged 
shock is more rapid and normal levels of blood 
pressure and cardiac output may usually be 
achieved afterward. 

Cardiac arrest is an all too frequent complica- 
tion of the operative treatment of patients with 
heart disease and frequently occurs during the 
induction of anesthesia. To investigate the mech- 
anism of cardiac arrest, which is usually attributa- 
ble to hypoxia, the systemic and cardiac circula- 
tion were separated in animals by means of two 
separate extracorporeal circulations. When nor- 
mal oxygenation of the heart was maintained and 
the body was rendered hypoxic, striking slowing 
of the heart rate was observed and cardiac arrest 
occurred in several animals. This study indicated 
that while the hypoxic heart is susceptible to 
arrest, the major cause of arrest is neurogenic 
stimuli which originate peripherally. The tech- 
nique of separating the peripheral and cardiac 
circulations has made possible other investiga- 
tions. When the temperature and oxygenation of 
the heart were kept normal and the body was 
cooled to 15° C. no significant changes in either 
the heart rate or its strength of contraction 
occurred. Only when the heart itself was cooled, 
and regardless of the general body temperature, 
were the usual myocardial changes which accom- 
pany hypothermia observed. The mechanism of 
action of angiotensin was also studied with this 
preparation. When the drug is given to an intact 
animal, striking elevations in blood pressure and 
contractile force occur. In the double perfusion 
system the effects of angiotensin on the heart and 
systemic circulation were separated. When the 
drug was given only to the heart no changes in 
blood pressure or heart rate occurred and there 
was actually a slight negative inotropic effect. 
When it was given into the peripheral circulation, 
however, a marked rise in blood pressure occurred 
immediately and later increases in pressure, heart 



rate, and contractile force were observed. These 
studies would indicate that angiotensin has no 
direct effect on the heart and that the increases 
in pressure and force which result from its ad- 
ministration occur as the result of peripheral sym- 
pathetic stimulation of the heart. 

Several projects, both clinical and experimental, 
have been carried out by the Anesthesia Research 
Unit, newly established in collaboration with the 
Department of Anesthesia, Clinical Center. The 
absolute and relative refractory periods of the 
heart and the diastolic electrical threshold of the 
heart have often been determined in experimental 
animals but never in man. In 20 patients, under- 
going cardiac operations, a stimulus-interval curve 
has been determined. Of greater importance have 
been studies of various drugs which can alter the 
stimulus-interval curve and the threshold of the 
heart to stimulation. It has been found, for exam- 
ple, that xylocaine has a striking effect in elevat- 
ing the electrical threshold of the heart without 
deleterious changes in contractile force or blood 
pressure. This is in contrast to the reactions 
observed with procaine amide, the drug most often 
utilized for the treatment of ventricular arrhyth- 
mia. Procaine has always been noted to cause a 
fall in blood pressure and contractile force and 
an actual decrease in the electrical threshold of 
the heart. These observations, combined with 
clinical observations in postoperative patients, 
indicate that xylocaine is the drug of choice in 
the treatment of ventricular arrhythmia and fur- 
nishes the physiologic background for its mech- 
anism of action. Further studies concern the 
effects of various anesthetic agents on the total 
volume of the venous system and the tone of the 
peripheral as well as the central veins of the body. 
These studies, when concluded, should provide 
information concerning some physiologic sequelae 
of the administration of various anesthetic agents. 

GERONTOLOGY BRANCH 

The research program of the Gerontology 
Branch is directed toward (1) identifying the bio- 
chemical, physiological and psychological changes 
that take place with increasing age in man, and 
(2) investigating the basic biological changes that 
influence aging in lower organisms in order to 
understand age-dependent alterations in the per- 
formance of humans. For the descriptive studies 
on age changes in the performance of humans, the 



120 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Branch has recruited a group of 500 males between 
the ages of 20 and 102 years who have volunteered 
to spend two days at the Baltimore City hospitals 
every 18 months to participate in a broad program 
which includes physiological and psychological 
tests as well as detailed medical examinations. 
These subjects live in their own homes in the com- 
munity and represent a highly educated and eco- 
nomically successful group. It is of great 
importance to determine the effects of age on the 
health and performance of subjects of this type in 
view of their importance to our society. In addi- 
tion, similar tests are conducted on older subjects 
who are admitted for custodial care in an Old 
People's Home. In connection with these studies, 
as well as those concerned with disease processes in 
the elderly, the Gerontology Branch staffs and 
operates a 40-bed ward at the Baltimore City 
hospitals. 

The program on the basic biology of aging 
seeks answers to questions such as : What changes 
occur in the enzyme activities of cells with the pas- 
sage of time? What is the biochemical basis of 
the increased life span of underfed animals? 
What are the mechanisms of cell death ? Do cells 
lose their capacity to divide with increased differ- 
entiation and the passage of time — or is the reduc- 
tion in cell division in the adult simply a reflection 
of alterations in the cellular environment? Is 
aging related to "rate of living"? What are the 
genetic factors influencing species differences in 
longevity ? Can aging effects be due to alterations 
in genetic information? Are somatic mutations 
an important factor in aging? Do molecules 
which are important in biological processes under- 
go age changes ? Is there formation of cross link- 
ages in proteins with aging in cells? Investiga- 
tions on these questions involve the use of a wide 
range of animal species such as the rat, mouse, 
hamster, Drosophila, rotifer, CampanulaHa, Eu- 
glena, etc., as well as cells growing and developing 
in tissue culture. In many instances it is neces- 
sary to extend knowledge of basic biochemical 
processes before age differences can be investigated 
and interpreted. Hence, part of the program of 
the Branch is devoted to basic biological prob- 
lems such as studies on the mechanisms of energy 
transformations in living cells and the structural 
characterises of biologically important com- 
pounds involved in these energy transformations, 



Aging in the Human 

The studies of age changes in physiological and 
psychological characteristics of a population of 
500 males, aged 20-102 years, who are highly edu- 
cated, successful and live in their own homes, have 
been extended by the completion of the second and 
third series of tests at 18-month intervals on part 
of the sample. A program for the transfer of ob- 
servational data to punch cards and magnetic tape 
for electronic computer analysis has been worked 
out and the medical history and physical examina- 
tions have been coded in preparation for punch- 
ing. This system has been designed with maxi- 
mum flexibility to permit a wide variety of sub- 

sequentanalyses ' 

Comparisons of Institutional and Community 
Residing Subjects 

Although insufficient time has elapsed since the 
beginning of the study to assess age changes in 
individual subjects, it has been possible to com- 
pare average values in the successful community 
residing group with those obtained on similar 
tests in a population residing in an institution for 
the aged, carefully selected to exclude subjects with 
clinical evidence of disease. In many perform- 
ance tests the average values obtained among com- 
munity residing subjects are significantly higher 
than those obtained for the institutional residing 
group. Examples of such performances include 
maximum breathing capacity, maximum work 
output of manual cranking and the ability to 
maintain a specified low level rate of manual work. 
In some of these functions, such as maximum 
breathing capacity, the average age decrement is 
significantly less in the community residing sub- 
jects than those residing in an institution. 

In contrast, a number of the physiological pro- 
cesses on which performance is based show no sig- 
nificant differences between the two groups in ab- 
solute values or age trends. Examples of such 
measurements include the strength of specific 
muscle groups, the total capacity of the lungs after 
adjustment for differences in body size, and the 
basal oxygen uptake based on the amount of metab- 
olizing tissues as estimated by intracellular water. 

These results lead to the conclusion that aging 
subjects living successfully in the community uti 
lize their failing physiological capacities more ef 



NATIONAL HEART INSTITUTE 



121 



fectively than do subjects who have resorted to 
congregate care. Thus, our conceptions about age 
changes in performance cannot be based on ob- 
servations made on institutionalized subjects 
alone. These results also emphasize the import- 
ance of sampling problems in drawing general 
conclusions about age changes in human perform- 
ance. 

Physiological Bases of Behavior 

Relationships between physiological and some 
behavioral characteristics were investigated in the 
community residing subjects. It was found that 
the variability of reaction time to an auditory 
signal increased significantly with age. This cor- 
relation between variability and age became in- 
significant when brain wave frequency was held 
constant by the use of partial correlation. It ap- 
pears that brain wave frequency is the central nerv- 
ous system factor concerned with age associated 
increases in variability as well as the mean value 
for response time. 

Using heart rate and amplitude of spinal reflex 
recorded during performance of the simple reac- 
tion time task as measures of arousal or activity 
level of the brain stem reticular system, two hypo- 
theses were investigated, viz, (1) that age is nega- 
tively correlated with level of arousal, and (2) 
that arousal and reaction time are related accord- 
ing to a U-shaped function. Neither hypothesis 
was substantiated by the data. 

Evidence that older individuals are less able to 
maintain attention in a repetitive and monotonous 
task than younger people has come to light in an 
investigation of watchkeeping behavior. In this 
study the subject is required to detect certain ir- 
regularities in the movement of a clock hand 
which occur only infrequently in the course of the 
task which lasts 1 hour. While the older subjects 
performed as well as the younger ones in the first 
15 minutes of the task, they failed to detect more 
of the irregular movements in the last 15 minutes 
of the test than the young did. 

Age Changes in Psychological Performance 

Previous experiments have shown that old sub- 
jects learn less effectively than young in an ex- 
perimental situation, but that when more time was 
given to select and make the response, the old sub- 
jects improved more than the yoimg. Another 
experiment has been performed in which subjects 



were paired on the basis of age and vocabulary 
scores. One member of each pair learned under 
conditions in which he could control the time taken 
to select and make each response ; the other mem- 
ber learned at the pace set by his partner. Under 
these conditions, pairs of young subjects showed 
no significant differences in learning performance, 
but in older subjects the self-paced member per- 
formed better than his partner. These observa- 
tions suggest that learning in older subjects is 
greatly favored where the individual can set his 
own pace. 

The Army Alpha Test of intelligence has been 
administered to subjects in the community resid- 
ing group. For young men aged 20-50 years the 
average age test score was 171 when the test was 
performed within a set time and 187 when the test 
was completed without time restriction. For older 
men aged 60-80 years the average score for timed 
performance was 151 and for completion without 
time limit was 181. Most older subjects in the 
sample have shown equivalent performance with 
their younger counterparts when time limits were 
removed although they perform less well in the 
time limited situation. 

Dietary Intakes 

Complete records of dietary intakes for seven 
consecutive days have been kept by subjects from 
the community residing group. These data will 
be used to identify age related differences in die- 
tary intakes and patterns of food selection in 
human subjects and to correlate differences with 
changes in anthropometric, physiological and bio- 
chemical measurements. A preliminary analysis 
of 7-day diet records submitted by 31 subjects has 
been made with respect to water, fiber, calories, 
protein, fat (saturated fatty acids, oleic and lin- 
oleic acids) carbohydrate, ascorbic acid, Ca, Fe, 
vitamin A, thiamine, niacin, and riboflavin. The 
data suggest that caloric intake and the percent of 
fat in the diet decline with age. This study will 
provide information, not now available, on the 
spontaneous eating habits of normal individuals 
where economic considerations are not a primary 
factor in determining foods eaten. 

Renal Physiology 

In addition to studies on overall performances 
in humans, other investigations have been focused 
on age changes in specific organ systems and 



122 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



physiological processes. Further evaluation of 
data obtained on community residing subjects dur- 
ing a 16-hour period of dehydration in the hos- 
pital indicates that the young subjects (age 20-39 
years) show the expected diurnal decrease in 
urine flow, total solute excretion and the increase 
in urine osmolality during the hours of sleep (12 
midnight to 6 a.m.) when compared to the waking 
hours (6 p.m. to midnight; 6 to 10 a.m.). The 
oldest subjects (age 70-100 years), carefully se- 
lected to exclude those with clinical signs of even 
mild edema or heart disease, showed a reversal of 
this pattern. Subjects of intermediate age (age 
40-69 years) showed a gradual shift in their urine 
excretion pattern toward that of the oldest 
subjects. 

The incidence of renal failure following surgi- 
cal procedures increases markedly with age. To 
assess the mechanism of this renal failure, clear- 
ances of inulin and paraaminohippurate (PAH) 
have been performed in a control preoperative 
period and 5 hours after operation in 36 patients 
over 50 years of age. Operations were of major 
types but not limited to one anatomic area. Con- 
trary to previous reports based primarily on ob- 
servations made on young subjects, significant 
changes in glomerular filtration rate (Cli) and 
renal blood flow (CIpah) were noted in the aged 
subjects. These findings throw new light on the 
relationship of both operative site and magnitude 
of the post-operative renal impairment. Results 
to date indicate: (1) following five small intra- 
abdominal operations, glomerular filtration rate 
increased slightly but plasma flow remained un- 
changed. Thus filtrate fraction rose. (2) Five 
thoracic operations (lobectomies and pneumonec- 
tomies) produced about the same changes in renal 
function. (3) Following extensive intraabdom- 
inal operations in 15 patients glomerular filtration 
and plasma flow decreased proportionally so that 
filtration fractions were not significantly changed. 
(4) In four patients with abdominal aneurysim 
resection there was a mean decrease in glomerular 
filtration rate and plasma flow postoperatively. 
These changes were not significantly different 
from the changes in group 3 (extensive intraab- 
dominal operations). Thus the nature of the 
changes in renal function appear to be related to 
the site and extent of the operative procedure. 



Endocrinology 

Recent observations show that acute febrile ill- 
ness causes a rapidly reversible but marked in- 
crease in thyroxine degradation rate without 
altering the concentration of circulating thy- 
roxine. This finding is most readily interpreted 
as indicating a homeostatic increase in thyroidal 
release of thyroxine in order to maintain a con- 
stant plasma level of the hormone. These measure- 
ments provide strong evidence that the thyroid, 
like the adrenal, participates rapidly in the re- 
sponse to at least some types of stress. Although 
only a few observations have thus far been made, 
elderly subjects seem to retain this homeostatic 
mechanism intact. If so, the slowed degradation 
seen in normal aged persons is not a fixed metabolic 
change and argues against an earlier proposal that 
the rate limiting step in thyroxine turnover is in 
the enzymatic degradation of the thyroid hor- 
mone. 

Work has progressed on development of a meth- 
od which may permit estimation of angiotension 
production rate in various states where the renin- 
angiotension-aldosterone mechanism is operative 
(unilateral renal ischemia with hypertension; sec- 
ondary aldosteronism) . The approach is to meas- 
ure the renal excretion of a dipeptide which is 
formed in the process of in vivo angiotensin ac- 
tivation. A method for measuring the peptide in 
urine is near completion and will soon be applied 
to the problem at hand. Incidental information 
on the heretofore unexplored area of the renal 
handling of certain peptides will become available 
in the course of this work. If successful, the 
approach should have general applicability in 
estimations of peptide hormone production rates. 

Carbohydrate Metabolism 

Previous experiments have shown that carbo- 
hydrate metabolism is impaired with increasing 
age. This is shown by the delayed disappearance 
of excess glucose from the blood after the adminis- 
tration of either an oral or an intravenous glucose 
load. Furthermore, the effects of cortisone on the 
rate of glucose disposition is greater in aged than 
in younger subjects. The impairment with age 
has also been demonstrated by the delayed rate of 
fall of the blood glucose concentration after in- 
travenous tolbutamide administration. These 



NATIONAL HEART INSTITUTE 



123 



tests are known to be stimuli to the release of 
endogenous insulin. Studies have therefore been 
started to test whether the age changes might be 
due to deficient insulin output by the pancreas 
in response to these stress tests or to deficient sen- 
sitivity of the tissues to insulin. Preliminary 
results in a group of aged subjects suggest that 
their peripheral tissues are indeed relatively in- 
sensitive to physiological concentrations of insulin, 
as tested by the forearm infra-arterial insulin 
tolerance technique. Yet the responses to in- 
travenous insulin tolerance tests (0.05 units/Kg. 
body weight) in these same subjects were generally 
normal. These apparently discordant results sug- 
gest that the hepatic sensitivity to insulin might 
be retained with age while skeletal muscle sen- 
sitivity decreases. It is also possible that the con- 
centration of insulin achieved in plasma in the 
intravenous test is so large that subtle age dif- 
ferences cannot be detected. Tests will therefore 
be repeated with smaller closes. 

These "impairments" in carbohydrate metab- 
olism with age raise challenging questions with 
regard to the diagnosis of diabetes mellitus. Ul- 
timately this diagnosis rests upon the finding of 
blood glucose concentrations which exceed certain 
standards under specified experimental conditions. 
These standards have generally been defined by 
studies on young subjects. If these commonly 
accepted standards are applied to older subjects 
an unacceptably high percentage of them (gen- 
erally more than 50%) would have to be classified 
as diabetic. Eeview of published reports reveals 
that the common solution to this problem is to 
raise the upper limit of normal to some level 
(completely arbitrarily) to achieve a more ac- 
ceptable percentage of abnormal results. The is- 
sue then is whether (1) standards for these tests 
need to be established with age as a variable or 
(2) diabetes is in reality an extremely common 
concomitant of aging. Studies have therefore been 
designed on the longitudinal subjects to charac- 
terize the commonly used tests for the diagnosis 
of diabetes. This group unquestionably will rep- 
resent the best control series available since, in 
other studies on aged subjects, the subjects, al- 
though generally "screened for the presence of 
metabolic diseases," are in reality chronically hos- 
pitalized or institutionalized individuals. As the 
data accumulate on the longitudinal subjects, cor- 
relation between their metabolic tests and certain 



diseases recognized as being concomitant or com- 
plications of the diabetic state will be examined. 
These analyses should provide an answer to the 
question of whether the high blood glucose con- 
centrations represent a physiological alteration 
with age or a true disease state. 

Biology of Aging 

Behavioral Changes With Age 

The purpose of this program is to determine 
the effects of age on various types of specific be- 
havior in the rat. The rat is a useful animal for 
these studies because of its use in many psycho- 
logical studies. Furthermore, the rat engages in 
a variety of activities for which quantitative in- 
dices can be devised. For example, exploratory 
behavior can be observed in the rat and can be 
distinguished from gross activity. The rat also 
engages in manipulative behavior which can be re- 
corded quantitatively. Other variables, such as 
light vs. dark, availability of food, the presence 
of other animals and the effects of previous ex- 
periences, can be experimentally varied. 

Preliminary experiments have shown that ex- 
ploratory behavior can be reliably measured. Tests 
have included barriers, multiple-unit intercon- 
nected "open fields" (enclosed spaces in which the 
animal is placed and observed) , "open fields" with 
and without hiding areas and small area "open 
field" tests of long duration. Animals were ob- 
served systematically at specific time intervals and 
behavior such as sniffing, grooming, or lying re- 
corded during a short time interval. The effects 
of dietary restriction on animals of different ages 
have been studied. Exploratory responses (snif- 
fing) increased with food deprivation while non- 
exploratory responses (lying or grooming) de- 
creased. Preliminary experiments indicate that 
age has an influence on exploratory behavior. In 
senescent animals, exploratory behavior decreases 
with increasing trials (experience) whereas it 
increases in young animals. Although explora- 
tory behavior is reduced in old animals, food de- 
privation results in a greater increase in explora- 
tory behavior in old than in young rats. Other 
factors such as experience and conditions of rear- 
ing (such as single or multiple caging) also play 
a role in exploratory behavior. Future experi- 
ments will attempt to determine the relative im- 
portance of these variables and their interactions. 






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ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



Effects of Nutrition and Parental Age on 
Longevity 

Restriction of food intake, beginning early in 
life is one of the few conditions which is known 
to increase life span in the rat. The restricted 
animals grow very slowly and for a longer time 
than normal rats. Hence it has been assumed that 
the increased longevity resulted from a reduc- 
tion in growth rate and that the retarded animals 
were physiologically younger than the normally 
ad libitum fed controls. Since the mechanisms 
of life extension in restricted rats is unknown, a 
research program has been instituted to try and 
define the biochemical basis of the phenomenon. 
In previously reported studies, the life expect- 
ancy of young rats was increased by reduced 
dietary intake and the concentrations of various 
enzymes determined at different times during the 
first year of life. Although there were demon- 
strable differences in selected enzymatic activities, 
for example, 35% increase in liver succinoxidase 
and 40% increase in kidney alkaline phosphatase, 
between these animals and normal ad libitum fed 
controls, they were not the same as those expected 
if the normal processes associated with growth 
had merely been retarded. Furthermore, during 
this year young growing animals were subjected 
to either 25, 50, or 75% reduction in food intake 
for 10 weeks. Measurements of the liver succin- 
oxidase have shown that the increments in the 
concentrations of the enzymatic activities were re- 
lated to the degree of restriction. On the other 
hand, although a decrease of 50 or 75% in food 
consumption increased the alkaline phosphatase of 
kidney 30%, a 25% reduction of food intake did 
not affect the concentration of the enzyme. Thus, 
it is apparent that quantitative differences in se- 
lected enzymes can be brought about by the degree 
of dietary restriction. This fact now makes it 
possible to determine whether longevity is related 
to the concentrations of these selected enzymes in 
rats subjected to dietary restrictions. More re- 
cent studies have indicated that the same changes 
in enzyme activities are brought about in adult 
animals subjected to reduced dietary intake. 
These data offer further evidence that the in- 
creased life span associated with dietary restric- 
tion is not the result of generalized retardation of 
normal processes but that dietary restriction in- 
duces changes in specific enzyme systems. On the 
basis of these findings, studies have been initiated 



to determine whether the life spans of adult (12 
month) and even senescent (19 month) rats can 
be increased by dietary restriction. Although 
these studies are still in progress, data obtained 
thus far suggest that the life span of adults but 
not of senescent rats may be increased by reduced 
dietary intake. This indicates that age plays a 
role in the response to changes in experimental 
conditions which affect longevity in young grow- 
ing animals. 

Since it has been found that dietary restriction 
in growing male rats produces an increased con- 
centration of renal alkaline phosphatase, the 
Gomori (cobalt sulfide) technique, modified to 
retain enzyme and approach zero-order kinetics 
during incubation, was applied to sections of kid- 
ney tissue in an attempt to localize the enzyme 
histochemically. Multiple incubation times were 
used. A "darkness index" was obtained by an 
objective, semi-quantitative evaluation of the 
slides, with the following results: (1) the alkaline 
phosphatase in restricted animals was confined 
almost entirely to the brush borders of the proxi- 
mal convoluted tubules; (2) the alkaline phospha- 
tase concentration within the brush border was 
increased in restricted animals; (3) the histo- 
chemical results correlated reasonably well with 
biochemical assays on the opposite whole kidneys 
in the same animals (r= 0.6053) ; (4) glomerular 
alkaline phosphatase was detectable after 8'-16' 
incubation times, but this was more pronounced in 
the ad libitum fed animals. 

Glomerular localization suggested a blood borne 
phosphatase, with reduced serum levels in restrict- 
ed animals. Serum levels were subsequently 
checked and a reduction of 20% was found in the 
restricted animals. 

Studies on the rotifer (Philodina citrina) have 
repeated the earlier work of Lansing who showed 
that the life span of progeny selected from old 
mothers is markedly shorter than that of the 
parent generation. However, the total egg pro- 
duction of the short-lived rotifers was significant- 
ly lower than that of the controls. If the 
decreased life span had resulted from a uniform 
acceleration of normal physiological processes, it 
would be expected that short-lived rotifers would 
produce the same number of eggs but in a shorter 
length of time compared to the longer-lived con- 
trols. Thus, the data obtained on both the rat and 
the rotifer suggest that experimental conditions 



NATIONAL HEART INSTITUTE 



125 



which alter life span are associated with some 
basic change in, rather than a retardation or accel- 
eration of, normal biochemical and physiological 
processes. 

The replacement of H 2 by D 2 in the nutrient 
medium has been foimd to increase significantly 
the larval pupal and adult lifetimes of fruit flies 
at concentrations of 10 and 20% D 2 in the 
nutrient medium. However, both larval and adult 
mortality is increased at concentrations of 60% 
D 2 0. Both development and reactions leading to 
aging thus appear to be slowed by moderate 
amounts of D 2 0. 

Effect of Environmental Temperature on Longev- 
ity 

According to one theory, aging and life span 
of a species is related to the rates of metabolic 
processes taking place in the animal. The obser- 
vation that life span in fruit flies can be extended 
by lowering the environmental temperature is 
offered as evidence for this theory. Experiments 
in our laboratory have shown that life span in 
the rotifer can also be extended significantly bj 
lowering the environmental temperature. Thus 
rotifers maintained at 6° C. lived longer (50% 
mortality at 98 days ) than those grown at 25° 
C. (50% mortality at 33 days). 

These experiments were extended to determine 
the effect of age at which rotifers were transferred 
to a cold environment on extension of the life span. 
Although 33% of the rotifers transferred to 6° C. 
at 24 days of age survived the longest lived con- 
trol (25° C) rotifer, the mean life spans of the 
two groups were not significantly different. How- 
ever, the per cent survivors at 98 days of rotifers 
transferred to 6° C. at 6, 12, and 18 clays of age 
were 55, 42, and 16% respectively. These results 
support data obtained on the rat that age does 
have an effect on the response of animals to experi- 
mental conditions which alter life span. 

In addition, biochemical studies on the rotifer 
are in progress and indicate that it is possible to 
obtain by sonication a preparation from this or- 
ganism which retains the activities of a variety of 
enzymes such as hexokinase, lactic dehydrogenase 
and glucose-6-phosphate dehydrogenase. Future 
experiments will be carried out to determine the 
effect of age on the activities of these enzymes in 
normal rotifers as well as those whose life span has 



been altered by either selection on the basis of 
parental age or exposure to cold. 

Effect of Radiation on Longevity 

The earlier observation that X-irradiation pro- 
longed the lifetime of fruit flies has been shown 
to be due to sterilization and preservation of the 
food supply. If the medium is replaced frequently 
the control (non-irradiated) flies live much longer 
and X-irradiation has only a life-shortening effect. 
Under these conditions 2,000 r is approximately 
equivalent to one day of aging. 

Aging in Cnidaria (Coelenterata) 

Representative organisms from four classes of 
the phylum Cnidaria (Coelenterata) have now 
been studied to determine the time dependence of 
death or regression. Both Campanularia and 
Bougainvillia exhibit a Gompertzian mortality 
curve after the first few days of development while 
a related colonial hydrazoan, Clytia, shows a mor- 
tality rate independent of age. Of a group of 45 
Cyanea capillata, an Anthazoan, no individuals 
were found to die in the 6-month period of obser- 
vation. The wide variations in rate and mode of 
senescence in this related group of organisms rec- 
ommends it for biochemical comparisons to reveal 
possible determinants of aging. 

Age Pigment 

While the nature and origin of the pigmented 
components of cardiac age pigment remain unde- 
fined, further studies of the isolated pigment indi- 
cate some similarities as well as differences from 
melanin and autoxidized lipid, two materials com- 
monly believed to be components of lipofuscin. 
The spectral distribution of the fluorescence of the 
extractable fluorescent components of age pigment, 
the Rf values on thin layer chromatography, as 
well as the insensitivity of the fluorescence to pH 
changes, clearly differentiates age pigment from 
the in vitro oxidation products of cephalin and 
fatty acids, or even the in vitro oxidation products 
of the extracted age pigment lipids themselves. On 
the other hand, the oxidation of unsaturated fatty 
acid esters in an ammonia atmosphere and the 
alkaline condensation of diacetyl, a suspected inter- 
mediate in fatty acid oxidation, have been found 
to yield a variety of fluorescent products, some of 
which seem similar to those of age pigment. 



126 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The elemental analysis of the hydrolysis-resist- 
ant polymeric fraction of cardiac lipofuscin indi- 
cates a nitrogen and sulfur content much too high 
for a simple autoxidized lipid. The nitrogen con- 
tent is lower and the hydrogen content much high- 
er, however, than that of natural melanins. The 
elemental composition is remarkably similar to 
that found by Moore and Wang for a hydrolysis- 
resistant pigment in vitamin E deficient rat muscle 
lending strength to the position that age pigment 
formation may involve lipid autoxidation. 

Analyses of the cardiac age pigment samples 
consistently indicate a distinctive amino acid com- 
position similar to that of some integument pro- 
teins although the high hydroxyproline content of 
collagen is lacking. Sonically isolated liver pig- 
ment shows considerable differences although 
glycine and proline are also high. Both analyses 
are entirely different from that reported by Sei- 
bert, et al. for cardiac lipofuscin isolated by a 
different method. We hope to resolve this dis- 
crepancy in the near future through exchange of 
samples. 

Electron micrography has confirmed that the 
isolated pigment particles are morphologically 
similar to the in situ pigment. There is a strong 
resemblance to particles described as liver lyso- 
somes by Essner and Novikoff. Although some- 
what less heterogeneous in appearance, the age 
pigment shows a similar osmophilia and absence 
of mitochondrial structural elements. A small 
contamination with mitochondrial fragments ob- 
served by electron microscopy may explain the 
cytochrome oxidase activity of the pigment prep- 
arations. 

Basic Biology 

In many instances investigations of the role of 
cellular processes in longevity and aging are 
hampered because of inadequate knowledge about 
the mechanisms involved. Hence, part of the re- 
search program of the Gerontology Branch is 
devoted to basic research in cellular biochemistry. 

Cell Division 

In many tissues, aging is associated with a grad- 
ual loss of functioning cells. This is especially 
true in tissues such as muscle and the nervous sys- 
tem where cell division is absent or minimal in the 
adult. Consequently, studies on the control of cell 



division in lower organisms may be of importance 
in the study of aging. 

The ability to induce synchronized cell division 
and growth in unicellular organisms by alternat- 
ing warm and cold temperature cycles has provided 
cells of uniform age. This homogeneity has made 
it possible to study certain factors regulating cell 
metabolism as well as the effects of antibiotics, 
sterols, and starvation on growth, cell division, and 
cell metabolism. 

In Astasia Tonga the TCA-soluble "metabolic 
pool" formed at the end of the warm and 
throughout the cold period. Addition of 8-aza- 
guanine during "pool" formation inhibits syn- 
chrony; addition after "pool" formation permits 
one synchronized cycle only. Thus 8-azaguanine 
inhibits substances which are synthesized during 
"pool" formation and which are necessary for 
normal mitosis, despite the low incorporation of 
this nucleic acid base analogue into cells. 

Cell division can be inhibited in Euglena grac- 
ilis by removing the source of either carbon or 
nitrogen from the nutrient medium. Thus, stable 
colonies with relatively constant cell number can 
be obtained which continue to live and metabolize 
for extended periods of time. A model of a meta- 
zoan can be obtained experimentally which per- 
mits the study of the effects of time on metabolic 
processes. 

A number of studies on metabolic processes in 
these stabilized (starved) colonies have been car- 
ried out. For example, the addition of actinomy- 
cin D to the culture medium (with refeeding) 
does not inhibit the lag period of growth, or pro- 
tein or DNA synthesis, and has only a small effect 
on UNA synthesis. Growth, however, is reduced 
and, although protein and DNA accumulate, RNA 
does not. Thus, despite starvation and the anti- 
biotic, the ribosomes evidently remain "pro- 
grammed". The effect of the antibiotic on RNA 
is not seen unless the culture is potentially capable 
of active division. 

Although acetate is considered to be the best 
carbon source for flagellate protozoa at neutral 
pH, both Euglena and Astasia reach population 
densities 10-15 times higher when grown on eth- 
anol than on acetate. 

During the period of exponential growth of 
Euglena. protein, RNA, and dry weight decline. 
Thus the often accepted, though rarely tested, con- 



NATIONAL HEART INSTITUTE 



127 



cept that in this phase of growth cells are in a uni- 
form state, must be revised. 

Oxidative Phosphorylation 

The ATP-dependent reduction of nicotinamide 
adenine dinucleotide (NAD or DPN) by succinate 
discovered in submitorchondrial particles has 
been examined in detail in continuation of the 
general aims of the project. The inhibition of the 
reaction by uncoupling agents including 2,4- 
dinitrophenol and by electron transport inhibitors 
(Amytal and malonate) leaves little doubt that 
the system represents the reversal of oxidative 
phosphorylation in the segment between NADH 
and cytochrome b. The assay offers a significant 
advantage over the previous systems based on in- 
corporation of phosphate into ATP since kinetic 
measurements can be readily carried out by fol- 
lowing the reduction of NAD spectrophotometri- 
cally. 

Since this system involves the succinic dehy- 
drogenase and the succinic-coenzyme (CoQ) re- 
ductase in addition to the DPNH-CoQ reductase, 
and only the latter should be theoretically neces- 
sary for the phosphorylation, attempts were made 
to simplify the system further. We now have 
what is perhaps the simplest and most rapid assay 
for oxidative phosphorylation reactions. It con- 
sists of measuring NAD reduction by ascorbate in 
the presence of menadione (vitamin K 3 ) or CoQ 
and ATP. The menadione is reduced non- 
enzymically by ascorbate and resulting hydro- 
quinone provides the reducing equivalents. ATP 
provides the "high energy" intermediates (via the 
coupling reactions of oxidative phosphorylation) 
that make the reaction thermodynamically possi- 
ble. The system may be represented probably as 
follows : 

Ascorbate + quinone^dehy dro-ascorbate 

+ hydroquinone (1) 

ATP + NAD<=>~NAD + ADP + Pi (2) 

~NAD + hyclroqumone?=±NADH 2 + quinone ( 3 ) 

Attempts at resolving the system into simpler 
components have been encouraging. A soluble en- 
zyme has been extracted which is essential for the 
ATP-dependent endergonic reduction of NAD by 
succinate or ascorbate and menadione. Since the 
particles, without the soluble enzyme, carry out 



the electron transfer in the thermodynamically 
favorable direction (viz, the oxidation of NADH 
by fumarate or quinone or reaction 3 from right 
to left) it is highly probable that the soluble factor 
represents the enzyme that catalyzes reaction 2 or 
the synthesis, in the reverse direction, of ATP 
from the primary "high-energy" intermediate in 
oxidative phosphorylation. 

Quite recently the NADH-menadione and 
NADH-CoQ reductases (reaction 3 from right to 
left) have been obtained in solution and are being- 
purified. We thus have in solution two compo- 
nents of the oxidative phosphorylation system. 
It may be pointed out that the solubilization of 
DPNH-CoQ reductase transport chain has not 
been reported before and has proved to be the 
largest single difficulty in studying the mechanism 
of oxidative phosphorylation. Our efforts are now 
directed towards purifying the two components 
and restoring the oxidative phosphorylation sys- 
tem in solution using other factors if necessary. 

Molecular Structure: DNA 

The major effort in the field of molecular struc- 
ture was devoted to the study of the various types 
of metal ion interaction with DNA and other 
polynucleotides. A large bulk of evidence has 
been accumulated to substantiate last year's pre- 
dictions, based upon effects of the metals on the 
melting out of DNA, that some metal ions bind 
to the phosphate and others to the nucleotide bases. 
The importance of this dichotomy is that, where- 
as the phosphate-binding ions are capable of main- 
taining the DNA structure intact, the base-binding 
metals bring about its denaturation. The latter 
effect may have physiological implications in that 
an accumulation of the base-binding metals could 
hinder the ability of the DNA to carry out some 
of its intended functions. 

The destabilization effect of the base-binding 
metals is of particular interest in the present study 
if the binding is base-specific, rather than random. 
The discovery that copper produces a batho- 
chromic shift in the ultraviolet spectrum of poly- 
inosinic acid, but not in the spectrum of the other 
polynucleotides, indicates that the binding is in- 
deed specific for guanine in DNA. In line with 
this interpretation, the destabilization effect of 



128 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



copper on DNA is greatest with DNA of the high- 
est guanine-cytosine content. These experiments 
suggest copper ion as an important reagent for the 
labelling of guanine on a polynucleotide chain. 

It has been demonstrated that lanthanum ion 
degrades all of the polyncleotides to low molecu- 
lar weight end products through the scission of 
phosphate bonds along the chain. The use of lan- 
thanum thus becomes another tool for the non- 
specific degradation of nucleic acids. It is hoped 
that the degradation can be rendered specific by 
the use of other reagents. 

Two such reagents that are specific for adenine 
are the nickel complex of dimethyl-sulfoxide and 
the copper complex of quinoline-carboxaldehyde. 
Methyl orange was found to react preferentially 
with cystosine. 

An effort has been made to utilize the base con- 
tents of the DNA of different invertebrate species 
in order to establish their phylogenetic relation- 
ships. Data were obtained on members of the 
phyla Cnidaria and Platyhelminthes. The data 
suggested the possibility of a relation between 
Hydra and Platyhelminthes, and are consistent 



with the hypothesis that Cnidaria and Platyhel- 
minthes have a common precursor. 

Deoxyribonuclease 

The effect of various metal ions upon the action 
of deoxyribonuclease was investigated. When the 
activity was plotted vs. metal ion concentration, 
the curves for all of the metals passed through a 
maximum, and the sequence of metals, listed in the 
order of concentrations at which they produce op- 
timal activity, followed the sequence of metal com- 
plex stabilities. Thus our last year's generaliza- 
tion to explain the variability of metal enzyme 
activation has been confirmed in the case of de- 
oxyribonuclease. 

Age Changes in Collagen 

Preliminary experiments show that solubiliza- 
tion techniques designed to preserve the structure 
of the native collagen aggregate produce maxi- 
mum solubility at the young adult (12 month) 
level. Periodate titration studies, which measure 
carbohydrate crosslinking, pass through a mini- 
mum at the same level. 



NATIONAL INSTITUTE OF ARTHRITIS AND 

METABOLIC DISEASES 



BASIC RESEARCH 



Introduction 



Perhaps the major events in the intramural 
scientific life of the National Institute of Arth- 
ritis and Metabolic Diseases have been the retire- 
ment of Dr. Floyd S. Daft, who served as Director 
of our Institute from 1953, and the resignation 
of Dr. DeWitt Stetten, Jr., who has served as 
Scientific Director since 1954. Fortunately, Dr. 
Daft has been able to remain here part time and 
continue his work on nutrition of germ-free ani- 
mals and also serve for advice and consultation. 
The consolation to be derived from the major loss 
of Dr. Stetten to our Institute is the knowledge 
that in his new position as Dean and founder of 
the Medical School of Rutgers University he can 
make an important contribution to medical educa- 
tion as well as medical research. Dr. Stetten's 
broad knowledge, foresight and stimulus will, 
however, continue as an influence in NIAMD. 
Further changes have been the resignation of Dr. 
Olaf Mickelsen from the Laboratory of Nutrition 
and Endocrinology and Dr. Heinz Specht from 
the Laboratory of Physical Biology. Their re- 
placements are Dr. John C. Keresztesy and Dr. 
John Buck. The long planned Laboratory of 
Molecular Biology is now fully and actively func- 
tioning with Dr. Gordon Tomkins as Chief. 

Honors have come to several of the scientists of 
this Institute. Dr. Joe H. Tjio received one of 
the new Kennedy Foundation awards for his work 
in isolation and characterization of human chro- 
mosomes, a work with an impact only now be- 
ginning to be felt. Dr. Harold Edelhoch received 
the Van Meter prize for his work on thyroglobulin. 

The work of the NIAMD is summarized in the 
following pages and a further summary here is 



hardly possible except to note the wide range of 
disciplines now represented and the broad inter- 
ests in this Institute. At its most theoretical end 
is the Laboratory of Mathematical Research, 
whose work is in part theoretical derivations of 
mathematical relationships relevant to biological 
and chemical problems and in part formulation 
of methods for the use of high speed computing 
devices for the solution of biomedical problems. 
There is then a gradation from theoretical chem- 
istry through physical chemistry to organic chem- 
istry where achievements have been outstanding 
in this Institute for many years. The discovery 
of new sugars of biological importance, of new 
methods of analysis of proteins, and of novel syn- 
thesis are reported. In molecular biology, a spec- 
trum of disciplines are used in exploring the 
mechanism of enzyme synthesis, of genetic con- 
trol, and of genetic transmission. Biochemistry 
is as usual well and productively represented in 
its activities. Vitamin metabolism and nutrition 
also continue to be a major interest of numerous 
of the Institute scientists. The broadly based 
clinical investigation program is dealt with sep- 
arately in this annual report. 

The Reserach and Clinical Associate programs 
are drawing some of the brightest and most in- 
quiring young physicians in the country and rep- 
resent not only a valuable educational service but 
reciprocally a yearly renewal of young talent for 
this Institute. It is not unreasonable to inquire 
about the other segment of medical investigators 
represented by those with the Ph.D. degree or 
those desirous of obtaining that degree. The 
young postdoctoral scientists now have a new pro- 
gram, the Staff Fellowship program, which per- 
mits an appointment of two or three years dura- 
tion, specifically designed for both their own de- 
velopment and our Institute's needs. 

129 



130 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



LABORATORY OF MOLECULAR BIOLOGY 

Investigations of Structure and Activity of Glu- 
tamic Dehydrogenase (Drs. G. M. Tomkins 
and K. L. Yielding) 

Investigations have continued on the relation of 
the structure to the activity of crystalline glutamic 
dehydrogenase. Further work using ultracentrif - 
ligation and light scattering have clearly indicated 
that the state of aggregation of the protein mole- 
cule does indeed influence its substrate specificity. 
Using light scattering, the influence of various re- 
agents on the molecular weight and enzymic ac- 
tivity of the enzyme has been investigated. 

Mechanism of Enzyme Induction in Mammals 

(Drs. L. D. Garren, S. Appel, R. R. Howell 
and G. M. Tomkins) 

It has been found that an increase in "messen- 
ger RNA" specific for several induced enzymes is 
produced when enzyme induction in mammals is 
stimulated upon the injection of steroid hormones. 
Indications have been obtained that there are two 
classes of "messenger RNA" in mammals — a stable 
unresponsive form and a labile form responsive to 
the presence of inducers. 

Mechanism of Enzyme Induction in Bacteria 

(Drs. D. Alpers, S. H. Barondes and G. M. 
Tomkins) 

A system of lysed protoplasts from E. coli 
which can form /?-galactosidase in the presence of 
inducer is being investigated. The system is in- 
hibited by actinomycin and puromycin and seems 
to have requirements for the various nucleotides 
and amino acids. The relation of this system to 
intact protoplasts is presently under investigation. 
It is hoped that the mechanism of induction of 
enzymes in bactei'ia will be elucidated by further 
studies using this preparation. 

Properties of the DNA-Dependent RNA Polym- 
erase From Mammalian Tissue (Drs. S. II. 
Barondes and G. M. Tomkins) 

The DNA-dependent RNA polymerase respon- 
sible for "messenger RNA" formation in mamma- 
lian tissue is being purified from liver nuclei. Its 
properties are under investigation and it is hoped 
that a study of this enzyme will yield results perti- 
nent to the problem of the regulation of "messen- 
ger RNA" synthesis in mammalian tissues. 



Active Transport of Histidine in Salmonella 

(Dr. G. Ferro-Luzzi Ames) 
A specific system for the transport of histidine 
in Salmonella typhimurium was shown to exist 
with a Km for histidine of 10 -7 M. A second per- 
mease for which the affinity of histidine is 10 -4 M 
is more general and responsible for the transport 
of aromatic amino acids tryptophane, phenylala- 
nine and tyrosine, as well as histidine, although 
for the latter three amino acids the affinity is some- 
what higher. Mutants resistant to aromatic amino 
acid analogs have been shown to be defective in the 
transport of aromatic amino acids. 

Effects of Natural and Synthetic Polyribonu- 
cleotides on Amino Acid Incorporation (Dr. 
E. S. Maxwell) 

The effects of natural and synthetic polyribo- 
nucleotides on amino acid incorporation into pro- 
tein were investigated in a cell-free system from 
rat liver. Isolated microsomal RNA stimulated 
the incorporation of all the amino acids tested, 
suggesting that mammalian microsomes contain 
some "messenger RNA". Soluble RNA, on the 
other hand, inhibited the incorporation. Using 
synthetic copolymers, RNA coding units were de- 
termined for 6 amino acids. Assuming triplet 
codes, the following compositions were indicated : 
phenylalanine, 3U; leucine, 2U1G or 2U1C; va- 
line, 2U1G; glycine, 1U2G; tryptophane, 1U2G; 
and serine, 2U1C. These results are in complete 
agreement with data reported for a system from 
E. coli. The fact that the same degeneracy is ob- 
served for leucine in the two systems seems of par- 
ticular interest. The data indicate that at least 
a portion of the code may be universal. 

Properties of Nucleic Acids and Their Compo- 
nents (Dr. H. T. Miles) 

Structural investigations on the nucleic acids 
have been carried out using infrared spectroscopy 
and the results have been correlated with other 
methods such as ultraviolet spectroscopy and op- 
tical rotation. It has been shown that the infrared 
is very sensitive to small changes in molecular 
structure and therefore possibly more sensitive to 
changes in configuration of complex macromole- 
cules such as the nucleic acids. Ordered structures 
involving a single strand of poly C with both 5' 
and deoxy 5' GMP have been studied, as well as 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



131 



the interaction of poly C with poly A and poly I. 
Studies of this type are of great interest in chem- 
istry of nucleic acids, as well as in understanding 
the possible biological role of these compounds. 

Chemical and Genetic Studies on Hemoglobin 

(Drs. H. A. Itano and A. Gottlieb) 

The genetic and chemical studies of inherited 
disorders of hemoglobin synthesis are being con- 
tinued. The chemical modifications of the arginyl 
residue of proteins is being investigated with the 
object of simplifying the analysis of tryptic 
hydrolyzates. 

Studies on Bacteriophage Lysozymes (Drs. W. 

Dreyer, T. Merigan and J. C. Bennett ) 

The lysozyme produced by E. coli on infection 
with bacteriophage T4 has been investigated by 
chemical, physical and enzymatic means. The 
amino acid sequence of the normal enzyme and 
certain mutant forms is under study; a number 
of mutants seem to produce a variety of altera- 
tions in amino acid sequence. It is presumed that 
the study of these mutants and a comparison of the 
enzyme produced by the wild type bacterial virus 
will be important in understanding the genetics of 
enzyme formation, amino acid interaction in- 
volved in stabilization of proteins and the nature 
of serologically reactive groups in the enzyme. 

Investigations on the Struciure of Guanylic Acid 

(Drs. D. R. Da vies, M. Gellert, M. Lipsett, 
H. T. Miles and P. Sigler) 

It has been found that concentrated solutions 
of 5' guanylic acid, a small molecule, exhibit all 
the properties of regular helical polynucleotides. 
Under suitable conditions, fibers can be obtained 
which give well oriented X-ray diffraction pat- 
terns which have been interpreted in terms of a 
specific model for hydrogen bonding between the 
bases. Qualitatively similar results have been ob- 
tained for 3' isomer of GMP as well. 

Kinetics of Interactions Between Synthetic Poly- 
nucleotides (Drs. D. R. Da vies, M. Gellert, 
M. Lipsett, H. T. Miles and P. Sigler) 

Reactions involving hydrogen bonding between 
strands of synthetic polyribonucleotides have 
been investigated. It has been demonstrated that 
these interactions are reversible and that their 
kinetics can be studied in the same way as those 



of simpler reactions. These studies are of consid- 
erable importance in understanding the mecha- 
nisms of synthesis and the reaction of the natural 
nucleic acids. 

X-Ray Diffraction Studies on Crystalline Chymo- 
trypsin (Drs. H. C. Skinner, C. Coulter, D. R. 
Davies and P. Sigler) 

In order to begin a full scale investigation on 
the structure of crystalline chymotrypsin, a search 
for a suitable heavy atom derivative of the enzyme 
has been made and computer programs for the 
analysis of collected data have been started. 

A Study of the Melting Point Dispersion Method 
Applied to Polynucleotide Structure (Drs. 
G. Felsenfeld, C. Smith and C. Stevens) 

This method, which allows the detection of se- 
lective melting out of AT or GC base pairs, has 
been applied to the problem of denatured DNA 
and of the structure of amino acid acceptor RNA. 
It has been shown that SRNA contains sequences 
of base pairs which are not random but consistent 
of runs of GC and AU base pairs. An additional 
feature of these studies is that an automatic data 
collection system has been designed which will 
facilitate the large numbers of experiments neces- 
sary for the application of this approach. 

Studies of Micrococcal Nuclease (Drs. G. Felsen- 
feld and P. von Hippel) 

It has been found that micrococcal nuclease is 
stabilized at high temperature by native but not 
denatured DNA. Thus, the ordered structure of 
the native nucleic acid interacts specifically with 
the protein. Since the enzyme can now be used 
at rather high temperatures, it is hoped that it will 
aid in the study of the early stages of nucleic acid 
degradation. 

Thermodynamics of Helix-Coil Transformations 

(Drs. P. Ross and G. Furukawa) 

It has been found that the helix-coil transition 
observed with DNA is accompanied by an anoma- 
lous heat capacity vs temperature plot. This per- 
mits the calculation of the standard enthalpy and 
entropy change accompanying the melting of 
DNA. This technique thus provides an approach 
to the determination of energy stabilizing ordered 
structure of nucleic acids and it could be extended 
to other macromolecules of bioloo-jcal interest. 



705-6S5— 63- 



-10 






132 



Biochemical Control Mechanisms in Histidine 
Biosynthesis (Dr. B. N. Ames) 

Further work has been done on the histidine 
operon — the cluster of 8 genes that makes the 
enzymes of histidine biosynthesis in Salmonella. 
Dr. Ames, in collaboration with F. Jacob and P. 
Hartman has investigated an operator mutant 
which had previousy been shown to cause all the 
histidine genes to be shut off even though they 
were not damaged by the mutation. It has been 
possible to mutate the operator mutant to new 
strains which have recovered the histidine en- 
zjmies. These strains have been found to be no 
longer under histidine repression control. One 
class of mutants has a deletion at the end of the 
histidine sequence, which suggests that the histi- 
dine genes have become part of a neighboring 
operon. The other class was found to have a 
duplicate histidine cluster translocated out of the 
chromosome : the duplicate piece is functional and 
not under histidine control. A number of other ex- 
periments also indicate that the 8 histidine genes 
function as a unit which can be turned on and off 
in response to the histidine repressor acting at an 
operator site in the gene at one extremity of the 
cluster — the gene that determines the sequence of 
phosphoribosyl-ATP pyrophosphorylase — the 
first enzyme of histidine biosynthesis. 

Dr. Martin has investigated feedback inhibition 
in the first enzyme of histidine biosynthesis. He 
has purified the enzyme and shown that there is 
a separate site on the enzyme for the histidine in- 
hibitor distinct from the substrate sites. This 
histidine site could be inactivated reversibly. 

LABORATORY OF BIOCHEMICAL PHARMA- 
COLOGY 

Spermine and Spermidine (Drs. H. Tabor, C. W. 
Tabor, U. Bachrach and L. deMeis) 

These amines are present in a variety of animal, 
plant and bacterial materials, and our studies have 
been directed towards their distribution, metab- 
olism and possible functions. 

During the past year our efforts have been 
mainly concerned with the enzymatic oxidation 
of these amines. Two enzyme systems have been 
studied : 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 

( 1 ) Serum amine oxidase 



NH 2 (CH2)3NH(CH 2 )4NH(CH2)3NH2+202+2H 2 



OHC(CH2)2NH(CH2)4NH(CH2)2CHO+2NH3+2H 2 



( 2 ) Serratia amine oxidase 

NH2(CH 2 ) ( NH(CH2)3NH 2 +02 ► NH 2 (CH 2 )3NH 2 +l ~+H 2 Oj 

Additional studies in this area during the past 
year have been ( 1 ) Development of a gas chromato- 
graphic assay for these amines, (2) Studies on 
the inhibition of phage growth by spermine, 

(3) Further studies on the concentration of the 
various amines in the pancreas of various animals, 

(4) Further studies with bacteriophage on the 
toxicity of the aldehyde formed by the action of 
serum amine oxidase on spermine, (5) Further 
studies on the effect of spermine in the stabiliza- 
tion of DNA against denaturation due to heating, 
(6) Further studies on the effect of spermine in 
pi'e venting shearing of DNA (with Dr. Dale 
Kaiser of Stanford University Medical School). 

Sialic Acids (Drs. L. Warren and R. Blacklow) 

The term sialic acid, refers to a group of com- 
pounds that are derivatives of neuraminic acid, 
a 9-carbon carbohydrate. They are usually found 
linked to various carbohydrate polymers, and, as 
indicated in part by the work summarized here, 
appear to be of importance in a variety of biologic 
areas. 

Further work has been carried out on the bio- 
synthesis of one representative of this group, N- 
acetylneuraminic acid. An enzyme has been puri- 
fied 125 fold from extracts of Neisseria meningi- 
tidis that carries out the following reaction : 
N-acetyl, D-mannosamine + phosphoenolpy ruvate 
— »N-acetylneuraminic acid + Pi 
The reaction differs from that found in mam- 
malian extracts which was described in last year's 
report (with Dr. H. Felsenfeld). 
N-acetyl, D-mannosamine-6-phosphate+ phospho- 

enolpyruvate— »N-acetylneuraminic acid-9-phos- 

phate + Pi 

Another reaction that has been described in 
extracts of N. meningitidis is : 

N-acetylneuraminic acid+CTP 
Mn 
— »CMP-N-acetylneuraminic acid + PP 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



133 



This reaction is of considerable potential impor- 
tance, since it may represent the activation of sialic 
acid for the synthesis of such mucopolysaccharides 
as capsular material, etc. 

An interesting and somewhat novel phase of the 
studies on sialic acids has been a study of their 
distribution in nature. Vertebrates and other 
chordates have sialic acids. These are also present 
in echinoderms, but not in tunicates. These find- 
ings have led to the suggestion that the usually- 
accepted theory of the tunicate origin of verte- 
brates is incomplete. In addition to its evolution- 
ary significance, these studies have led to the 
discovery of several new members of the sialic 
acid group , including a methoxy-derivative in 
starfish. 

Biochemistry of Sulfur-Containing Compounds 

(Dr. S. Black) 

It has been found that yeast glutathione reduc- 
tase is a flavine-adenine-dinuckotide-containing 
flavoprotein with -SH groups sensitive to -SH 
reagents only in the presence of reduced pyridine 
nucleotide. Because of these properties the en- 
zyme becomes an especially interesting subject for 
mechanism studies. To make such investigations 
feasible, methods have been sought to obtain puri- 
fied enzyme in adequate quantity. Several new 
procedures have been developed based upon the 
enzyme's surprisingly high stability in acid solu- 
tion and its adaptability to purification by ion-ex- 
changing chromatography. The best preparations 
thus far have a minimum molecular weight of 
about 400,000 based on FAD content, and a turn- 
over number of 8,000. It is hoped that homoge- 
neous material will be available in the near future. 

Turnover and Intracellular Distribution of 
Amino Acids in Bacteria (Drs. E. Levine and 
Loretta Leive) 

The formation of certain adaptive enzymes in 
Bacillus sub tills requires the presence of trace 
amounts of amino acids. This is not true in 
Escherichia coli cultures. Studies have demon- 
strated that the rate of protein turnover in B. 
subtil is is approximately y 10 of that in E. coli, and 
presumably this accounts for the above observa- 
tions. As part of these studies an apparatus was 
developed to distinguish intracellular turnover 
from intercellular turnover due to cell death and 
autolysis. This apparatus, which permits the equi- 



libration of culture fluids of separated bacterial 
suspensions, will be of general use in studies of 
turnover in bacteria. 

In other studies differences were observed in the 
utilization of labeled exogenous diaminopimelic 
acid by the bacterial cell for two different reac- 
tions; i.e., (1) incorporation of diaminopimelic 
acid into cell-wall precursor and (2) decarboxyla- 
tion to lysine for protein synthesis. It has been 
shown that this is the result of heterogeneity in 
the distribution of the added amino acid within 
the cell. In addition, the data indicate spatial 
separation of the two enzymes of diaminopimelic 
acid utilization. These results are important since 
they show that the intracellular environment is 
not an undifferentiated pool of metabolites, but is 
inhomogeneous both with respect to enzyme loca- 
tion and metabolite distribution (with Dr. B. 
Davis of Harvard Medical School). 

Control Mechanisms for Enzyme Levels in Mam- 
malian Systems (Dr. B. Schimke) 

Considerable work has been carried out in 
various laboratories on the factors controlling the 
level of enzyme in bacterial systems. Comparable 
control mechanisms are present in mammalian 
systems. These have been shown in (a) whole 
animal experiments and (b) tissue culture studies. 

The whole animal experiments represent a con- 
tinuation of the work done last year, and show 
that in the rat each of the enzymes involved in 
urea synthesis (carbamylphosphate synthetase, 
ornithine transcarbamylase, argininosuccinate 
synthetase and cleavage enzymes, and arginase) 
increases when protein increases and decreases 
when protein intake decreases. A particularly im- 
portant aspect of this work is the demonstration 
that the enzyme level is regulated by active con- 
trol of both enzyme synthesis and of enzyme 
degradation. 

Tissue culture studies have also been carried out 
on arginine metabolism, and demonstrate phenom- 
ena comparable to repression. For example, low 
arginine concentrations in the medium result in a 
marked increase in the level of argininosuccinate 
synthetase and a decrease in the level of arginase. 
Contrariwise, high arginine concentrations in the 
media have opposite effects. 

An important development during the work was 
the demonstration of the importance of using 
PPLO-f ree cells for the tissue culture studies. In 



134 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the presence of PPLO, arginine was rapidly de- 
graded by arginine deaminase, coupled with orni- 
thine transcarbamylase, and carbamyl phospho- 
kinase present in the PPLO. Many cell-lines 
commonly used are PPLO-contaminated (with 
Dr. M. F. Barile of the Division of Biologic 
Standards) . 

Metabolism of Histidine, Histamine and Related 
Imidazoles and Amines (Drs. C. McEwen 
and H. Bauer) 

Further studies have been carried out on the 
enzyme, diamine oxidase, which carries out the 
oxidative diamination of both histamine and a 
variety of diamines. We have developed a num- 
ber of new assays for diamine oxidase, but the 
most satisfactory assay developed has been a modi- 
fication of a previous method based on the colored 
quinolinium compound formed when the oxida- 
tion product of putrescine, A'-pyrroline, reacts 
with o-aminobenzaldehyde. 

This assay has been applied to the measurement 
of diamine oxidase in sera from pregnant woman, 
and is considerably more convenient than the bio- 
logical assays used for this purpose in the past. 
In pregnancy the diamine oxidase of the serum 
progressively rises, and is many times higher than 
the levels in non-pregnant controls. 

Diamine oxidase has been further purified from 
hog kidney with a 500 fold overall purification. 
Spectrophotometric studies on the purified enzyme 
have demonstrated a substrate reducible peak at 
about 500m/i. The cofactors of this enzyme are 
not known yet, and further work is being carried 
out on this aspect of the problem. Continuing at- 
tempts are being made to synthesize imidazole- 
acetaldehyde, the probable product of histamine 
oxidation. 

Studies on Burns (Drs. R. C. Millican, K. Mark- 
ley and S. M. Rosenthal) 

Studies were continued on the factors involved 
in the acute and delayed mortality following a 
high-temperature scald burn of the leg in mice. 
Septicemia was observed during the shock period, 
which was reduced by antibiotics. It seems likely 
that this septicemia is caused by invasion through 
the gut wall by the normal intestinal flora. 

Animals which had survived a previous scald of 
one hind leg demonstrated a decreased acute and 
late mortality when scalded on the other hind leg. 



as compared with the controls which had not been 
previously scalded. These experiments show that 
mice can acquire resistance to repeated scalding. 
This resistance can also be transferred passively 
as reported last year when convalescent scald 
plasma was found to protect animals against a 
severe scald. 

Studies have been continued in both the labora- 
tory and in a clinical study (Lima, Peru) on the 
Pseudomonas infections that occur in late deaths 
in burns. This is a very important problem in 
burn therapy since the development of these in- 
fections is usually fatal. In the laboratory, anti- 
gens have been prepared from various Pseudo- 
monas strains isolated from clinical cases for use 
in typing studies (with Dr. Ronald Wood of Johns 
Hopkins) . 

As part of these studies on Pseudomonas or- 
ganisms, the biosynthesis of pyocyanine was stud- 
ied ; this is the pigment formed by Pseudomonas 
aeruginosa. Pyocyanine was shown to be derived 
from C 14 -shikimic acid. 

Mevalonic Acid (Dr. K. Markley) 

Studies in this area have been directed towards 
discovering new pathways for mevalonic acid uti- 
lization. As part of these studies, Lactobacillus 
acidophilus has been grown on C 14 -mevalonic acid, 
and a large incorporation of label has been demon- 
strated in an unidentified neutral lipid, contain- 
ing a free hydroxyl group, that does not appear 
to be one of the common steroids. Glucose has 
been found closely associated with the radioactiv- 
ity, and further studies are in progress on the 
identification of the lipid-glucose complex. 

Leprosy (Dr. Y. T. Chang) 

The chemotherapeutic studies on mouse leprosy 
that have been conducted for many years are con- 
tinuing, but have now been supplemented with 
chemotherapeutic studies on human leprosy. For 
the latter studies, the technique of Dr. Charles 
Shepard on the growth of these organisms in mouse 
footpads has been used. Definite suppressive ac- 
tivity in mouse leprosy was observed with etham- 
butol, vadrine, sulfamethoxypyridazine, and sul- 
famonomethoxine. Streptomycin, B663, and di- 
aminodiphenylsulfone were very effective against 
the human leprosy organism in the footpad experi- 
ment (with Dr. C. Shepard, CDC, Atlanta). 

Continued improvements have been attained in 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



135 



the culture of Mycobacterium leprae murium. The 
growth of this organism in macrophages in tissue 
culture represents a particularly important con- 
tribution to these studies. Only preliminary 
studies (with Dr. Richard Adler of NIAID) have 
been carried out with human 31. leprae; healthy 
bacilli were observed in cultures of mouse perito- 
neal macrophages. 

Effect of Fasting on Fatty-Changes in Mice (Dr. 
S. H. Webster) 

Studies of the triglyceride content of liver, kid- 
ney, and heart showed that this increased after 
fasting. Fasting also resulted in marked increases 
in glutamic-oxalacetic transaminase and lactic de- 
hydrogenase ; this increase was not found in mice 
pretreated with dibenzyline (with Dr. B. Highman 
of the Laboratory of Experimental Pathology) . 

LABORATORY OF NUTRITION AND ENDO- 
CRINOLOGY 

Vitamin E, Antioxidants and Selenium 

The metabolic relationship between vitamin E 
and selenium has not been clearly elucidated. 
Earlier studies from this laboratory showed that 
one probable function of selenium is to increase 
the antioxidant capacity of tissues as does vitamin 
E. Continuing studies have revealed another simi- 
lar physiological effect produced by dietary vita- 
min E or sodium selenite. It has been known for 
many years that the vitamin effectively protects 
animals against certain types of chemical poisons. 
It has now been found that in chicks trace amounts 
of selenium have a similar action. Dietary levels 
of selenite which are below the usual requirement 
effectively prevent the toxicity and death which 
result when relatively high levels of certain organic 
compounds are fed. 

Another relationship between these two nutri- 
ents, and also to cystine, has been demonstrated in 
their effect on coenzyme A levels in chick liver. 
When chicks were fed purified diets with either 15 
or 25 percent of protein, minus the usual supple- 
mental cystine, coenzyme A in liver was reduced 
by one-third. Addition of vitamin E or selenite 
to the diet resulted in normal concentrations of the 
coenzyme. 

The fate of a-tocopherol (vitamin E) in the body 
has received only limited attention. In experi- 
ments with rats and chicks in which a-tocopherol 



labeled with C 14 was given in a single oral dose, 
it was found that from 35-50 percent was excreted 
in the feces, with no change in the compound, in a 
21 clay period. The uptake by different organs 
varied considerably, with liver, lungs, spleen, kid- 
ney and blood having from 1-5% of the dose after 
24 hours. Elimination from most of the tissues 
was rapid and by 21 days only lungs and small in- 
testine had as much as 0.5 percent of the dose. All 
radio-activity was accounted for as unchanged a- 
tocopherol, with no evidence for metabolic inter- 
mediates or end-products (Drs. J. Bieri and S. 
Krishnamurthy) . 

Protein Deprivation and Energy Metabolism 

This continuing study is designed to determine 
how variation in the protein status of animals af- 
fects the energy utilizing mechanism in liver cells. 
Individual enzymes, and cofactors, which partici- 
pate in the transfoi'mation of energy have been 
measured while rats are gradually depleted of 
protein, and also while they are being repleted 
with protein. It has been found that the liver 
loses an appreciable number of cells during pro- 
tein depletion but that cell reproduction proceeds 
rapidly when protein is re-fed. Methionine in the 
protein-free ration significantly reduced the loss 
of liver cells. 

In protein deficient rats not receiving methio- 
nine, the efficiency of energy utilization was con- 
siderably better than that in similar rats given 
methionine, even though the livers from the latter 
animals had a much higher rate of oxidation of 
succinate. The livers of the protein-depleted ani- 
mals had considerable losses of succinic dehydro- 
genase and also of cytochrome oxidase. 

About 80% of the liver coenzyme Q was lost 
during severe protein depletion. Following the 
repletion of protein, liver coenzyme Q, initially re- 
bounded to above normal levels and then returned 
to normal after about eight weeks (Dr. J. N. 
Williams, Jr.). 

Lipid Metabolism 

This investigation is directed toward a bio- 
chemical explanation for the abnormal lipid me- 
tabolism in rats which result from feeding 1% of 
orotic acid, a metabolic precursor of pyrimidines 
which occurs in milk. The large accumulation of 
fat in the liver which orotic acid produces has 
been found to be independent of wide variations in 



136 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the dietary protein or fat levels. The liver lipid 
has been characterized as almost all triglyceride 
containing only palmitic, palmitoleic and oleic 
acids. Cholesterol in these livers increased 5-6 
fold. Serum triglycerides fell almost to zero in 
orotic-fed rats, and cholesterol, phospholipid and 
unesterified fatty acids were reduced to 20% of 
normal. 

By labeling the body water with tritium, it has 
been shown that orotic acid causes an increase in 
fat synthesis in all tissues, and that it is this newly 
synthesized fat which accumulates in liver. 

Orotic acid isotopically labeled in various po- 
sitions is rapidly oxidized to carbon dioxide after 
its injection into rats ; it is converted, however, to 
pyrimidine nucleotides before being catabolized. 
Addition of adenine to the diet alters the path- 
way of metabolism of orotic acid (Dr. H. G. 
Windmueller) . 

Guinea Pig Nutrition 

The best purified diet suitable for experimental 
studies with the guinea pig was previously devised 
by Dr. Reid. This diet, however, does not produce 
the optima] growth obtainable with a commercial 
ration made of natural feedstuffs. It has been 
found that if 20% of the casein in the purified diet 
is replaced with an isonitrogenous amount of 
alfalfa, growth now becomes optimal. Studies are 
currently attempting to determine what compo- 
nent of alfalfa gives this growth response. 

Contrary to what occurs with other species, it 
has been found that the D and L-isomers of meth- 
ionine are not equally effective in the guinea pig. 
It is not clear whether this is due entirely to an 
actual metabolic difference, or whether variations 
in the dietary protein or possible changes in in- 
testinal flora may be involved (Dr. M. E. Reid). 

Diabetes and Fat Metabolism 

The effect of insulin on fatty acid synthesis in 
pancreatectomized rats was studied by measuring 
incorporation of tritium labeled body water into 
carcass and liver fatty acids. Pancreatectomy had 
no effect on fatty acid synthesis in fasting rats. 
The rate of synthesis in fed pancreatectomized 
rats, however, was 80% less than that in fed nor- 
mal rats ; insulin restored the rate to normal. In 
contrast to findings made in alloxan diabetic rats, 
insulin deprivation for 48 hours did not alter the 
responsiveness to insulin. In vitro studies made 



with liver slices and adipose tissues from the above 
animals, measuring conversion of glucose and 
fructose to fatty acids, confirmed the in vivo 
studies. 

The high rate of fatty acid release by incubated 
adipose tissue from pancreatectomized rats de- 
prived of insulin for 48 hours was markedly re- 
duced by in vitro addition of a small amount of in- 
sulin. Glucose uptake by incubated adipose tissue 
of untreated diabetic rats was normal. However, 
conversion of glucose to C0 2 , glyceride-glycerol 
and fatty acids was markedly depressed, while 
lactate formation was greatly increased. Utiliza- 
tion of glucose was normalized by addition of 
insulin. 

Dexamethasone at very low concentrations, 10~ 8 
to 10^ 7 M, accelerated release of fatty acid by in- 
cubated adipose tissue from normal fasted rats. 
Corticosterone produced similar effects if ten 
times as much was added. 2-a-methyl Cortisol 
was as potent as corticosterone, whereas 2-a- 
methyl cortisone was ineffective. Deoxycorticos- 
terone had little effect on fatty acid l'elease. Dexa- 
methasone also decreased uptake and metabolism 
of labeled glucose to carbon dioxide, total lipid, 
and fatty acid. Addition of small amounts of 
insulin to the media (4 mU/mill) reversed the ef- 
fects of dexamethasone on adipose tissue. These 
findings support the hypothesis that development 
of ketosis, fatty liver, and hyperlipemia in the dia- 
betic rat is the result of a direct action of the glu- 
cocorticoid on adipose tissue (Dr. R. O. Scow, Dr. 
S. S. Chernick and Dr. J. N. Fain) . 

Two aspects of fat formation were investigated 
in the liver: Lipogenesis, the formation of new 
fatty acids; and esterogenesis, the formation of 
new glyceryl ester bonds, as in triglycerides and 
phospholipids. Esterogenesis may involve esteri- 
fication of free fatty acids or transfer of fatty 
acids from one glyceride-linkage to another. In 
vitro incorporation of glycerol, formed from the 
metabolism of C 14 glucose or fructose, into tri- 
glycerides and phospholipids by liver slices was 
found to be extensive and independent of the rate 
of lipogenesis. This finding supports the previous 
observation that the perfused liver readily trans- 
ferred fatty acids of triglycerides to phospholipids 
and other triglycerides without the appearance of 
appreciable amounts of free fatty acids in blood 
or tissue (Dr. S. S. Chernick) . 

In perfusion studies adipose tissue removed tri- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



137 



glycerides intact from the circulation. The tri- 
glycerides were then hydrolyzed, extravascularly, 
to glycerol and fatty acids by lipoprotein lipase. 
The resultant fatty acids were re-esterified, prob- 
ably within the fat cells, to triglycerides or were 
released to the circulation. 

Fat cells were isolated from adipose tissue by 
treatment with collagenase. All of the lipopro- 
tein lipase in the tissue was found associated with 
the fat cells. The isolated fat cells appeared to 
be viable since they released free fatty acids when 
incubated with epinephrine or ACTH and utilized 
glucose at a faster rate when incubated with in- 
sulin (Dr. M. Rodbell and Dr. R. O. Scow) . 

Protein Hormones 

Growth of the body and various organs were 
studied in rats bearing transplantable pituitary 
tumors (MtT). Previous studies had shown that 
these tumors produce relatively large amounts of 
prolactin, growth hormone, and adrenocortico- 
trophic hormone. The weights of the heart, liver, 
kidney, spleen, preputial, and mammary glands 
were greatly increased in tumor bearing rats, 
whereas the body weight and length were only 
slightly greater than that in normal rats. The 
weights of the pituitary, pancreas, ovary, and 
uterus were less than normal while the thymus and 
white fat were completely atrophied. Adrenalec- 
tomy greatly increased body weight and weight 
of the tumor, thymus, and preputial gland in 
tumor bearing rats. The liver, kidney, and heart, 
however, were normal in weight. Thyroidectomy 
prevented the gain in body weight, gut length, and 
weight of the tumor and preputial gland in rats 
bearing tumors but did not prevent hypertrophy 
of the viscera. 

The changes in organ weights seen in tumor 
bearing rats were reproduced in normal rats by 
injecting a mixture of prolactin, growth hormone, 
and ACTH in very large doses. When the hor- 
mones were given singly, ACTH was the only one 
that produced hypertrophy of the heart and 
kidney. 

The systemic bioassay for prolactin has been 
greatly improved by using adult instead of juve- 
nile pigeons and injecting for seven instead of 
four days (Dr. R. W. Bates and S. Milkovic). 

Thyroid stimulating hormone (TSH) has been 
isolated from several species of animal, including 



human. Amino acid analysis has revealed differ- 
ences in their composition, principally with re- 
spect to cystine, glycine, lysine, and the amino 
sugar, galactosamine. Differences in electropho- 
retic mobility and biological and immunological 
properties can be related to differences in amino 
acid composition. Eel TSH was found to con- 
tain only half as much cystine as does the hor- 
mone from other species. The ineffectiveness of 
this preparation in the thyroid gland of higher 
vertebrates may be clue to a less highly organized 
tertiary structure as well as differences in primary 
structure. 

Density gradient centrifugation studies have 
shown that all preparations have the same sedi- 
mentation rate. TSH in blood of mice bearing 
TSH-producing tumors can be readily separated 
from the major protein fractions of plasma by 
density gradient centrifugation and by gel filtra- 
tion. Thus, it appears that the hormone in blood 
is not associated with /J or y globulins, as reported 
by other laboratories. 

Antibodies prepared against purified human 
TSH are highly specific for human TSH and have 
little cross-reactivity with bovine TSH. This casts 
doubt on the usefulness of anti-bovine TSH in 
the measurement of human TSH in blood. 

Examination of freshly prepared bovine TSH 
by molecular sieving on sephadex has revealed 
that purification can be increased beyond that 
achieved by ion-exchange chromatography. This 
finding suggests that the natural form of the hor- 
mone is more potent than any isolated so far (Drs. 
Condliffe and Y. Fontaine). 

Recent studies have established the purity of 
parathyroid hormone obtained as a homogeneous 
polypeptide. The methods used included starch 
gel electrophoresis, ultracentrifugal analysis of 
the hormone in guanidine using interference op- 
tics, and recovery of N-terminal alanine. Untra- 
centrifugal studies have shown that pure parathy- 
roid hormone forms high-molecular weight aggre- 
gates in certain buffers. This probably accounts 
for separation of pure preparations into three 
fractions with ammonium sulfate and for altera- 
tion of elution patterns on gel columns with 
changes of buffer conditions. Now that these 
properties have been defined, it is possible to pro- 
ceed with structural analysis of parathyroid hor- 
mone (Dr. G. Aurbach and Dr. J. T, Potts, NHI) . 



138 

Folic Acid 

An investigation of the folic acid profile in 
chicken liver extracts, on DEAE-celluose col- 
umns revealed the presence of a variety of mono- 
glntamate and polyglutamate derivatives. Using 
the analytical procedures developed in this labora- 
tory the monogiutamates identified include the 
N 10 -formyl-,N°-formyl- and the N 5 -methyl deriva- 
tive of tetrahydrofolic acid. A spectrum of N 10 - 
formyl- and N 5 -methyl- polyglutamyl derivatives 
of reduced folic acid of varying complexity was 
also observed. From this it appears that no single 
simple method of assay for the important vitamin 
cofactor in tissue is available at this time. 

The overall concentration of folic acid deriva- 
tives in tissue and the significance of the relative 
concentrations of the various forms of folic acid 
is a major problem and is being continued (Drs. 
Silverman and Noronha) . 

While enzymatic oxidation of 5-methyltetrahy- 
drofolate (PrefolicA) yields 5,10-methylenetetra- 
hydrofolate, the product of the initial chemical 
oxidation was a dihydro-derivative. This com- 
pound, shown to be 5-methyl-5,6-dihydrofolate, 
was readily reduced back to the tetrahydro- level 
by ascorbic acid, /?-mercoptoethanol and homo- 
cysteine. However, attempts to reduce this com- 
pound enzjanatically were unsuccessful. In ad- 
dition, attempts to oxidize the methyl group of 
this derivative to the methylene level were also 
unsuccessful (Dr. K. O. Donaldson and Dr. J. C. 
Keresztesy). 

Attempts to purify chemically prepared 5,10- 
methylenetetrahyclrofolic acid by gradient chro- 
matography on DEAE or TEAE-cellulose col- 
umns using carbonate buffer (pH 9.5) resulted in 
the separation of the two diasterioisomers of this 
derivative. One was identified as the biologically 
active isomer which corresponds to the 1,L isomer. 
The other was shown to be biologically inert and 
corresponds to the d,L isomer. However, both 
forms could be reduced to the methyl level by 
chemical reduction. This procedure provides a 
simple method for the preparation of fully active 
derivatives at the reduced forms of folic acid (Dr. 
B. Kaufman, Dr. K. Donaldson and Dr. J. C. 
Keresztesy ) . 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Large-Scale Laboratory 

The large-scale laboratory has continued to in- 
crease the help it gives to investigators through- 
out the whole National Institutes of Health. The 
greatest number of requests have come for grow- 
ing non-pathogenic organisms and bacterio-phage 
in the 100-gallon fermenter, to be used as sources 
of enzymes, DNA and RNA, in protein synthesis 
studied. The low temperature concentrating fa- 
cilities were found to be extremely useful for proc- 
essing heat-sensitive biological materials (Dr, 
J. C. Keresztesy and Dr. D. L. Rogersoii, Jr.). 

Germ-Free Program 

Work in the germ-free animal area has pro- 
gressed along several lines during the past year. 

It has been established by comparison of germ- 
free and conventional animals that a "normal" 
bacterial flora contributes folic acid to the host 
animal. A portion is obtained by the rat by in- 
gestion of feces but when coprophagy is com- 
pletely prevented by affixing tail cups to the ani- 
mal sufficient folic acid is obtained, undoubtedly 
by direct absorption, to prevent the appearance of 
frank deficiency (E. C. McDaniel, Dr. F. S. Daft, 
Dr. L. G. Herman [DRS], and R. G. Horn). 

Under certain conditions (when large amounts 
of vitamin C or certain antibiotics are added to a 
deficient diet) the conventional rat requires no 
dietary pantothenic acid. Under these conditions 
the vitamin synthesized by gastrointestinal bac- 
teria is made available to the host animal in suffi- 
cient amounts to meet the animal's needs. It has 
been established, however, that this phenomenon 
depends on coprophagy. It is still possible that 
the rat obtains some pantothenic acid by direct 
absorption without coprophagy but, if so, in 
amounts far below the animal's needs (E. G. Mc- 
Daniel, Dr. F. S. Daft, Dr. L. G. Herman [DRS], 
and R. G. Horn). 

Dietary liver cirrhosis clue to a deficiency of 
choline and related compounds has been postu- 
lated by other investigators to depend on the ac- 
tion of bacteria on a liver weakened by dietary 
insult. It has been established, however, that 
germ-free rats and mice develop dietary liver cir- 
rhosis with an incidence and severity probably 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



139 



equal to or greater than conventional animals 
(Dr. S. M. Levenson [Walter Reed Army Medi- 
cal Center], E. G. McDaniel, and Dr. F. S. Daft). 

Dietary liver necrosis on the other hand is fav- 
orably influenced by the germ-free state. Germ- 
free rats grow more rapidly and develop liver 
necrosis more slowly and in lower incidence than 
their conventional or conventionalized counter- 
parts. The basis of the deleterious influence of 
gastrointestinal bacteria on this condition is under 
investigation (E. G. McDaniel, Dr. F. S. Daft, 
Dr. L. C. Herman [DRS], and R. G. Horn). 

It has been shown by Pearson, a grantee, that 
arthritis can be induced in conventional rats by 
the injection of Freund's adjuvant. The question 
naturally arose as to the possible participation of 
infection in the development of this pathological 
condition. In collaborative studies it has been 
shown that this phenomenon occurs in "germ- 
free" animals, thus excluding the participation, as 
far as we have been able to determine, of all bac- 
teria. Still further studies will be carried out, 
however, in an attempt to exclude — or implicate — 
any extremely fastidious bacteria, whose presence 
might possibly have been missed in the tests which 
have been performed (Dr. C. M. Pearson 
[UCLA], Dr. F. D. Wood [UCLA], E. G. Mc- 
Daniel, Dr. F. S. Daft, and Dr. L. G. Herman 
[DRS]). 

LABORATORY OF PHYSICAL BIOLOGY 

As the lineal descendent of the Industrial 
Hygiene Research Laboratory, the Laboratory of 
Physical Biology is one of the oldest of NIH, as 
well as one of the largest. As such it illustrates 
the diversification of scientific activities that in- 
evitably develops over the years when independent 
investigators are free to explore subjects of their 
own choice and to follow where experiment leads. 
Even so, it has been surprising to find that a few 
major research themes encompas nearly all the 
work of the Laboratory. In the following eclectic 
summary of current LPB findings and interests 
in these primary areas, only the work of full 
time professional members of the Laboratory are 
mentioned. In the reporting, an effort has been 
made to omit technical details and to simplify the 
presentation so that it will be more accessible to the 
non-specialist. 



Physiology 

This not ideal heading is used to include the 
multifarious LPB projects in which intact organ- 
isms or cells are studied with one or more of the 
following objectives in mind: (a) Elucidating 
some normal function, (b) observing mechanisms 
of adaptation or compensation to stress, (c) un- 
raveling changes occurring during growth and dif- 
ferentiation. For example, tolerance to altitude 
in rats has been found to be lower in males than 
in non-pregnant females, and lower at puberty 
and in old age than in middle age (Altland). 
Curiously, 18-20 hours of altitude hypoxia enables 
rats to avoid the. edema ordinarily caused by in- 
travenous dextran, and prior intraperitoneal hy- 
pertonic glucose has a similar protective effect, 
shown not to be osmotic (Marshall). 

In continuing work on the complex problem of 
what induces somatic cells of the simple metazoan 
Hydra to differentiate into gonad tissue, it has 
been found that constant stirring of the culture 
medium inhibits sexuality in sparse populations 
but not in more crowded cultures (suggesting the 
presence of a soluble endogenous inducer) and that 
low temperature promotes sexuality in one species 
but inhibits it in another (Park) . A superficially 
similar inverse temperature effect on the relative 
infectivities of Trypanosoma cruzi and Ldish- 
mania enrietti is being studied in relation to re- 
spiratory metabolism and the integrity of the mito- 
chondrionlike kinetoplast (Greenblatt). 

A literature report of a specialized C0 3 -elimi- 
natiner mechanism in certain insects was found 
to be groundless, but new data on the respiratory 
system was obtained as a byproduct (Keister). 
In another investigation on insects, involving the 
effects of temperature on respiratory rate during 
different stages of development, an interesting by- 
product was the discovery of cold-inactivation of 
the hormone that induces pupation (Burkett). 

Molecular Structure 

In addition to much work on macromolecules 
(see below), LPB chemists and physicists have 
put great effort into elucidating the structure, 
properties and reactions of simple molecules 
which can form building blocks of biologically im- 
portant substances or yield clues to their construe- 



140 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tion. For example, periodicities in electron mi- 
crographs of calcium stearate crystals have been 
studied to assess certain diffraction effects caused 
by molecular lattices which have to be understood 
in order to interpret pictures of protein crystals 
(Labaw). Similarly, a paracrystalline state of 
protein fibers has been implicated as the basis of 
the transparency of the lens of the eye (Trokel). 
Studies on the vibrational spectra of quinone de- 
rivatives (Becker, Charney) have an obvious bear- 
ing on bonding in quinoid molecules in general 
and in many complex natural products in particu- 
lar. Hydrogen bonding, of great importance in 
protein structure, has also been studied intensively 
by Dr. Becker and collaborators. 

Among physical methods of studying molecular 
structure, optical rotatory dispersion has certain 
unique capabilities. By applying this tool to non- 
planar cis and trans dienes it has been possible 
to confirm theoretical predictions of the configura- 
tions and conformations of such molecules and to 
elucidate the structure of Erythrina alkaloids, 
compounds which have a curare-like action 
(Weiss, Ziffer, Charney). 

Complexes of metal ions with organic molecules 
such as amino acids (chelates) have been studied 
from several angles. From nuclear magnetic res- 
onance spectra the binding sites and rates of ex- 
change of cupric and cadmium ions with several 
glycyl peptides have been established (Li, Shein- 
blatt, Becker) , while ultraviolet spectroscopy has 
shed light on the association of mercury with 
amine and imide groups of nucleosides, in relation 
to the mercurial disruption of the nucleic acid helix 
(Simpson). Still other methods were applied to 
the study of binding of inorganic ions (Na + , H + , 
Ca ++ ) to B-lactoglobulin, ribonuclease and serum 
albumin (Baker, Saroff, Carroll, Wolff), the re- 
sults of which are important in relation to protein 
structure. 

On the analytical side, both cis and trans iso- 
mers of a neAv amino acid from sponge collagen 
and from the antibiotic telomycin (3-hydroxy-L- 
proline) and a new di-peptide from iris (a-L- 
glutamyl-/?-alanine) have been isolated (Irre- 
verre) and one of the bright red, photosensitizing 
pigments of the mold Elsinoe has been purified and 
structurally characterized (Weiss, Batterham). 
Progress continues on the adaptation of gas chro- 
matography to amino acid analysis, using the 
methyl and trifluoroacetylmethyl esters (Nicholls, 



Makisumi, Saroff) and esterification has also been 
used successfully in the gas chromatography of 
the organic acids of the Krebs cycle (Sharpless). 

Protein Structure, Activity and Synthesis 

LPB work on macromolecular structure has 
centered on the proteins of muscle and of blood 
clotting, and on certain enzymes. The tropomyo- 
sins of larval and adult insects have been found 
to show only minor differences (Kominz, Maru- 
yama, Levenbook). Configurational changes in 
succinylated myosin have helped elucidate the 
mechanism of polymerization and dissociation of 
this muscle enzyme and its cleavage at the disul- 
fide bond (Kominz, Nihei). Similarly, bacterial 
dehydrogenases are being studied in an effort to 
understand their variations in molecular aggrega- 
tion (Carroll). 

Certain polypeptides which appear during the 
clotting of fibrinogen and which have the interest- 
ing property of potentiating the bradykinin- 
induced contraction of smooth muscle, are under 
investigation, as is sialic acid, a carbohydrate 
moiety that is lost during the clotting process 
(Laki, Gladner, Osbahr, Irreverre, Chandrasek- 
har) . Bovine thrombin has been found to be an 
unusually small enzyme (62 residues: m.w. ca. 
8000) and its polymerization and amnio acid se- 
quence are being studied (Gladner, Osbahr, Laki, 
Carroll). 

Biosynthesis of protein is being studied analyti- 
cally in the favorable situation of insect metamor- 
phosis (Levenbook, Shigematsu), using the en- 
zyme aldolase as the test molecule. In the yeast 
Candida utilis various purine and pyrimidine 
analogs have been shown to influence both distri- 
bution and total quantity of both endogenous and 
exogenous amino acids in an "internal pool" and 
studies of the incorporation of labeled amino acids 
have revealed fallacies in a standard method of 
assessing amino acid turnover (Kempner). 

Biological Energy Transduction and Coupling 

A major LPB effort is going into the very inter- 
esting and promising domain which concerns the 
biological machinery for transforming one type of 
energy into another or of triggering the release of 
one type of energy by another. The absorption 
of light quanta, for example, may lead to electri- 
cal signals in nerve (which are membrane phe- 
nomena) that eventuate in vision, or to endother- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



141 



mic chemical combination (photosynthesis). 
Conversely, chemical changes may lead to light 
production (bioluminescence), with or without 
the mediation of triggering by nervous impulses, 
or may result in cellular intake or expulsion of 
material against concentration gradients (active 
transport) . Similarly, bioelectric events may set 
off chemical reactions which lead to folding of 
protein chains (muscular contraction). 

Insight into mechanisms by which pigments 
trap light was gained by the demonstration that 
reactivity of excited diazoacetophenone molecules 
can be related to the electronic configuration of 
the ground state (Ziffer, Sharpless). Investiga- 
tion on one photosensitizing pigment ("Elsino- 
chrome A") have already been mentioned 
(Weiss) , and it has been shown that the ability of 
the chlorophyll molecule to absorb energy in the 
far red depends on its specific physical orientation 
in the chloroplast (Olson, Jennings). Also, in 
an attempt to determine the quantum yield of 
rhodopsin bleaching, retinine isomers are be- 
ing separated by thin-layer chromatography 
(Adams). 

By the use of a novel method for measuring si- 
multaneously the absorption spectrum and electri- 
cal activity of thin slices of living retina it has 
been shown that a sequence of chemical reactions 
occurs, transforming the highly oriented pigment 
rhodopsin into the bleached, unoriented alkaline 
metarhodopsin. The onset of receptor action cur- 
rent coincides with the final step (Hagins, 
Srebro). The relation between chemical and bio- 
electric events has also been studied in two other 
neuroeffector systems, the firefly light organ and 
striated muscle. In the former, flashing has been 
shown to be invariably associated with character- 
istic volleys of nerve spikes and numerous other 
details of central and peripheral modulation and 
excitation have been reported (Buck). In living 
myofibrils activated by micro-application of Ca ++ , 
the dependence of contractility on sarcomere 
length indicates that myofilament interdigitation, 
rather than peculiarities of the activation system, 
controls the sarcomere length at which contractil- 
ity vanishes (Podolsky). 

In glycerinated muscle fibers, energy transfer 
from ATP in relation to hydrolysis products and 
ionic strength were investigated, and the kinetics 
of ATP diffusion into muscle have been studied 
intensively, both empirically and theoretically 



(Bowen) . Studies are continuing on the relation- 
ship of the actin, actin-myosin and actin-tropomy- 
osin complexes to muscidar contraction via de- 
tailed physical-chemical studies (Kominz, Maru- 
yama, Laki). 

Most, if not all, excitation and activation proc- 
esses in living cells involve changes in or at bio- 
logical membraness. It is therefore most relevant 
that some recently developed liquid membranes 
were shown to have an extreme degree of ionic se- 
lectivity, being readily permeable to anions and 
virtually impermeable to cations (or vice versa), 
with relative rates of the two permeation processes 
up to 100,000 :1 or better. These membranes also 
show a considerable degree of ionic specificity, 
thus opening up interesting vistas for the prob- 
lem of ionic specificity in living membranes, car- 
rier mechanisms, and ion-binding in general 
(Shean and Sollner). Similarly, the action of 
drugs and hormones on surface films of precisely 
known composition is yielding quantitative data 
on both permeability and structural alterations of 
interfaces (Gershfeld). 

In conclusion it may be of interest to mention 
that various members of LPB are beginning to ex- 
plore the potentialities of computer analysis of 
complex data, for example spectrophotometric ab- 
sorption curves of steroids (Brackett, Hahn, 
Sharpless), nuclear magnetic resonance spectra 
(Becker, Bradley) and in the computation of mo- 
lecular weights, ion binding curves and amino acid 
sequences (Shapiro), Computation Branch, NTH. 

LABORATORY OF CHEMISTRY 

Rotatory Dispersion of Nucleosides 

As a first step in a study of the causes of the 
remarkable optical rotatory anomalies shown by 
certain pyrrolidine deoxynucleosides the rotatory 
behavior of a wide variety of 2-deoxy-D-ribose 
derivatives has been examined by Drs. Ness and 
Bhattacharya. Where oxygen or halogen is at- 
tached to carbon one of this sugar, rotational be- 
havoir appears to be completely normal. Atten- 
tion is now being turned to various nitrogen gly- 
cosides bearing special relationships to the nucleo- 
sides. 

Cis-Nucleosides 

Conventional methods for the synthesis of nu- 
cleosides (other than 2-deoxynucleosides) are suit- 



142 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



able only for the preparation of nucleosides where- 
in the aglycon bears a trans relationship to the 
hydroxyl at carbon two of the sugar moiety. Gis 
isomers are needed for optical rotational studies 
as well as for studies in the chemotherapy of can- 
cer. Through the use of 2,3,5-tri-O-benzyl-D- 
arabinofuranosyl chloride a cis nucleoside, 9-/?- 
D-arabinofuranosyladenine ("spongoadenosine") 
has been synthesized in good yield by Dr. C. 
Glaudemans. 

Octuloses and Nonuloses 

The study of the higher-carbon sugars in avo- 
cado and Sedum species was continued. The struc- 
ture of the second octulose from the avocado was 
proved by Drs. Sephton and Kichtmyer to bs D- 
glycero-~L-galacto-octu\ose by degradation with 
lead tetraacetate aaid with oxygen in alkaline 
solution, and was confirmed by synthesis, both en- 
zymatic and chemical. The structure of the first 
nonulose from the avocado, previously established 
by degradations as D-erythro-Ij-gluco-noxmlose, 
was confirmed by chemical synthesis. Although 
aldoheptoses have been reported previously as 
constituents of bacterial polysaccharides, the isola- 
tion of D-glycero-'D-galacto-heptose from the 
avocado marks the first known appearance of a 
heptose in the plant world. 

Anhydroheptuloses 

About five years ago a quantity of Li-galacto- 
heptulose was transformed by hot dilute acid to 
give about a 5% yield of nonreducing anhydro 
sugars. Separation of the mixture on a Dowex-1 
column (borate form) led to the isolation of about 
equal amounts of two products whose structures 
have now been established by Mr. Zissis as 2,7- 
anhydro-a-Jj- galacto heptulofuranose and 2,7- 
anhydro-/?-L-#aZacto-heptulopyranose by the ap- 
plication of methods used earlier for similar com- 
pounds. 

Neighboring Group Rearrangements of Cyclitols 

Continuation of studies on the behavior of car- 
bohydrates in liquid hydrogen fluoride has shown 
that cyclitols and 1,5-anhydrogiycitols as such are 
unattacked but that their esters smoothly undergo 
Walden inversion at the central carbon atom of 
a cis-tram triacycloxy sequence. It is obvious that, 
this experimentally simple process shows great 
promise for the synthesis of difficultly accessible 



organic structures. As an example, the work in 
the cyclitol series demonstrated that the hexaace- 
tate of the common form of inositol (??iy<?-inositol) 
is readily converted to «me<9-inositol, hitherto one 
of the least accessible cyclitols. 

Glycosyl Cyanides 

The increasing use of mercuric cyanide as an 
acid acceptor in the Koenigs-Knorr synthesis of 
glycosides and disaccharicles makes the side reac- 
tion, the condensation of mercuric cyanide with 
acylated glycosyl halides, a matter of immediate 
importance. Dr. B. Coxon has shown that 2,3,4,6- 
tetra-<9-acetyl-a - D - glucosyl - pyranosyl bromide 
condenses with mercuric cyanide to give two prod- 
ucts, the expected 2,3,4,6-tetra-(9-acetyl-/?-D-glu- 
copyranosyl cyanide and the highly novel struc- 
ture 3,4,6 - tri - O - acetyl - 1,2 - - (1 - cyano- 
ethylidene) - a - D-glucopyranose. The glycosyl 
cyanides are readily preparable and offer interest- 
ing intermediates for the synthesis of C-glycosides 
related to pseudouracil. 

Progress on Benzomorphans 

The chemistry, pharmacology and clinical 
studies on this new series of centrally acting 
agents have progressed to the point where, (1) 
structural limitations are fairly well defined; (2) 
relatively accurate predictions may be made re- 
garding transfer of results from animal to man; 
(3) the /2-series of 5,9-dialkyl-6,7-benzomorphans 
(5-alkyl quasiequatorial, 9-alkyl axial for the hy- 
clroaromatic ring, i.e. oriented toward nitrogen) 
corresponding to the unnatural morphine configu- 
ration at the /?:C ring junction are 10-60 times 
more potent than the more accessible a-isomers and 
morphine and not commensurately more toxic or 
addictive; (4) it can be stated beyond reasonable 
doubt that a sharp, consistent separation of strong, 
central analgesic activity from addiction liability 
and other adverse effects (morphine toxiphrenia) 
has been achieved for the first time; (5) a parallel 
between analgesic and conditioned response-block- 
ing activity is observed in animals. Of the three 
2' - hydroxy - 5 - alkyl - 2 - methyl - 6,7 - benzo- 
morphans studied to date, 5-ethyl and 5-propyl are 
much better than 5-methyl and are comparable to 
morphine (levo) even as the racemates. With the 
5,9-dialkyl compounds maximum activity is shown 
in both the a- and ^-series when the sum of the 
carbons of the two C-alkyl groups is 3. Activity 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



143 



does not fall sharply until the carbons of these two 
groups totals 6 (dipropyl). Especial attention is 
directed to the following 2'-hydroxy-2-methyl-6,7- 
benzomorphans as candidates for further study, 
(a) (±)-5-Ethyl and 5-propyl, ED 50 2.3 and 2.1 
mg./kg. (analgesic activity, mouse) ; physical de- 
pendence capacity (PDC, monkey) none in single- 
dose suppression studies up to 30 mg./kg. Mor- 
phine has ED 50 2.1 and is rated high in PDC with 
a stabilizing (abstinence suppression) does of 3 
mg./kg. (b) ( — ) -5,9-Dimethyl, ED 50 1.7, analgesic 
activity in man comparable to morphine, PDC 
(monkey) low, abstinence suppression capacity in 
man one-eighth that of morphine, capacity for pro- 
ducing physical dependence in man less than that 
of morphine, (c) a-(±)-5,9-Diethyl, ED 50 4.1, 
nearly equivalent to morphine in relieving pain in 
man, PDC in monkey none to 60 mg./kg. (single- 
dose suppression), low in 30-day chronic adminis- 
tration compared with high for morphine; acute 
toxocity very low. (d) /?- ( ± ) -5,9-Diethyl, ED 50 
0.28, PDC none to 12 mg./kg.; acute toxicity 
(mice) low. 

Role of Phenolic Hydroxyl in a- and /3-Benzo- 
morphans 

The analgesically favorable effect of the phe- 
nolic hydroxyl in structures with a heterocyclic 
tertiary nitrogen has been known for some time. 
However, the role of this substituent in tolerance 
and physical dependence has not hitherto been 
studied because deoxy compounds of the order of 
potency of morphine are not known. The high 
activity of the aforementioned benzomorphan 
/3-diastereomers provided optimism that deoxy 
compounds of morphine-like potency were not im- 
plausible. Consequently, a- and /3-5,9-diethyl-2- 
methyl-6,7-ben2;omorphans have been synthesized 
by two different routes and their stereochemistry 
confirmed by methiodide-formation-rate experi- 
ments and by conversion to known compounds. 
Preliminary evaluation indicates that the a-com- 
pound (devoid of oxygen) is indeed nearly as ac- 
tive as morphine and therefore suitable for the 
further research mentioned above. The ^-counter- 
part expected to be more potent than the a, is how- 
ever, much less so and constitutes the first excep- 



tion in the benzomorphan series in this respect 
(Arthur E. Jacobson). 

Approach to /?-Benzomorphans, the More Potent 
Series 

Further attempts to improve the yields of the 
pharmacologically more interesting /3-benzomor- 
phan diastereoisomers of the benzomorphan series 
have been successful. Thus, cyclization of the hy- 
drochloride or hydrobromide salt of 2-benzyl- and 
2 - (p - hydroxy benzyl) - 3,4 - dialkyl - 1 - methyl- 
1,2,5,6 - tetrahydro - pyridines with aluminum 
bromide or chloride has afforded 40-50% yields of 
the /^-isomers in 3 :1 and 2 :1 predominance over a 
in contrast to 48% HBr at 140° which favors a 
10:1 and 85% H 3 P0 4 (180°) which gives 50% 
yields of a and 10-15% of £ (J. H. Ager, E. M. 
Fry, S. E. Fullerton, E. L. May). After a com- 
plex series of rearrangements and chemical manip- 
ulations with 1,2-dihydro-pyridines it has been 
possible to devise a feasible synthesis of trans-2- 
benzyl-l,3,4-trimethyl - 1,2,3,6-tetrahydropyridine 
hydrobromide (I) which can be cyclized to /?-2,5, 
9-trimethyl-6,7-benzomorphan in 95% yield or to 
almost exclusively the a-congener with 48% HBr. 
Since it was also shown that I was readily re- 
arranged to the corresponding A 3 -piperidene by 
strong acid one may postulate that the formation 
of the /^-compounds from the A 3 -piperidenes may 
be preceded by rearrangement to I and related A 4 - 
piperidenes and that a-compounds are produced 
principally from A 3 -piperidenes (E. M. Fry). 

Structure-Activity Studies in the Codeinone 
Series 

Efforts to determine the effect on analgesic ac- 
tivity, of altering the point of attachment of ni- 
trogen from carbon 9 to carbon 7 in dihydrocode- 
inone have continued. The difficulty of fission of 
the oxide ring during dry distillation of an inter- 
mediate quaternary bromide has been circum- 
vented by performing the pyrolysis (for methyl 
bromide extrusion) in 1-heptanol. Quantities of 
the new tertiary base are being prepared for car- 
bonyl reactions with organo-metallic reagents. 
The stereochemistry of these reactions and prod- 
ucts as well as the pharmacological effects of such 



144 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



substitutions will be emphasized (L. J. Sargent 
and B. C. Joshi). 

Preliminary Screening of Analgesics 

Preliminary evaluation (mice) for analgesic 
activity and toxicity of new compounds coming 
principally from outside sources continues. Ex- 
ploratory experiments on 78 substances, complete 
assays on 71 compounds and 150 toxicity experi- 
ments have been conducted (E. L. Atwell and J. 
Goodwin), and the data analyzed by statistical 
(probit) analysis (W. Ness). 

Morphine Tolerance Studies 

Study of morphine tolerance development as a 
possible immune response phenomenon was con- 
ducted by comparison of the analgesic response 
(rat, hot plate) of animals treated with a combina- 
tion of Freund's adjuvent and morphine with that 
of animals treated with morphine alone. Adju- 
vant-treated animals retained sensitivity to mor- 
phine several months longer than those treated 
with morphine alone (J. Cochin and J. Goodwin). 

Rapid Test for Addiction 

Thin-layer chromatography analysis of urine 
for narcotics and metabolites is rapid and sensi- 
tive, complements the Nalline test in the quick de- 
tection of addiction, and may be important to 
prompt treatment of barbiturate overdosage. Sev- 
eral new metabolites have been uncovered by this 
technique. Thin layer chromatography has also 
been developed for estimation of other toxic ma- 
terials as antihistamines, tranquilizers, etc. in 
blood and tissues. Many clinical and criminolog- 
ical laboratories have adopted these procedures. 
(J. Cochin and John Daly). 

Synthetic Antigens 

Polyethyleneimine-amino acid polymers were 
fabricated and haptenic groups introduced. Sev- 
eral of these are being tested by Dr. Sehon of 
Canada (T. D. Perrine) for the synthesis of po- 
tential antigens containing a strictly hydrocarbon 
backbone with built-in, judiciously-spaced sugar 
moieties as determinant groups, practicable syn- 
theses for p-vinyl-phenyl /?-glucoside, maltoside 
and cellobioside have been worked out. These gly- 
cosides were purified and characterized extensive- 
ly for the subsequent polymerization reactions 
(Lionel Clingman, T. D. Perrine) 



Chemistry of Narcotine 

A research program involving transformations 
of the plentiful, relatively non-toxic, little-used 
opium alkaloid narcotine has been initiated, with 
a view to exploring the pharmacologic possibilities 
of derivatives of this versatile chemical. LiAlH 4 
reduction gave the known diol but the Grignard 
reaction gave not the reported unsaturated com- 
pound, but due to a milder isolation procedure the 
precursor carbinol (A. E. Jacobson). 

Quinazoline Glucosides as Potential 
Carcinostatics 

Because of its overall steric resemblance to 
purine, quinazoline (as an unnatural base) was 
selected for nucleoside and nucleotide studies more 
particularly for elaboration of abnormal nucleo- 
sides and ultimate incorporation into small syn- 
thetic nucleotides. These base derivatives might 
be expected to exert biological (carcinostatic) 
effect by inhibition of certain key enzymes (thy- 
midylate synthetase) or by acting as purine antag- 
onists. They might also be effective as general 
antiviral agents. Quinazolinedione-mercury, 2,4- 
diethoxyquinazoline and their tetrahydro counter- 
parts were severally condensed with acetobromo- 
glucose to give three new, acetylated glucosides. 
Attempts to deacetylate these substances resulted 
in unexpected cleavage to the initial base and glu- 
cose suggesting that O- rather than N-glucosides 
were formed. The good possibility of rearrang- 
ing these O— »N glucosides is now being considered. 

Active Center of Ribonuclease 

The revised sequence of ribonuclease, amino acid 
residues 11-18, has been published jointly by the 
group at the Rockefeller Institute, the Laboratory 
of Cellular Physiology and Metalbolism of the 
NHI and by the Laboratory of Chemistry, 
NIAMD. Dr. Erhard Gross of the latter group 
has extended these studies to the non-anzymatic 
cleavage of the methionine bond of the so-called 
S-peptide, the 20-unit peptide obtainable from 
ribonuclease by the action of Nagarse. The hep- 
tapeptide 13-20 was purified and characterized on 
ion exchange columns where it appeared as a dou- 
ble or triple peak depending on subtle differences 
in the pH of the citrate buffer. Simultaneously 
there was investigated a synthetic heptapeptide 
prepared in the laboratory of Prof. K. Hofmann 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



145 



which also gave rise to one or two corresponding 
peaks on ion exchange chromatography. It is sus- 
pected that the striking sensitivity or subtle 
changes in pH of the "natural" as well as the syn- 
thetic heptapeptide may reflect in some degree a 
lability which is part of the intrinsic property of 
the active center of ribonuclease. It is well known 
that the S-peptide and the ribonuclease core re- 
combine to form an active enzyme. It will now 
be possible to study the recombination of the core 
with the two S-peptide fragments, namely, the 13- 
unit peptide and the heptapeptide and to look 
for reappearance of enzymatic activity. 

These findings have stimulated further applica- 
tions. It may well be that peptide sequences that 
are part of the active center or are associated with 
the mode of action of certain enzymes will not 
exhibit unusual properties such as conformational, 
configurational or structural tautomerism or la- 
bility until they have been peeled out of their 
tertiary structure casing. After the loss of stabi- 
lization by hydrogen bridges the active center in 
situ or in nuce may then show lability phenomena 
accessible to investigation by physico-chemical 
methods. So far trypsin has been cleaved into 
three fragments by cyanogen bromide, whereas 
any fragments resulting from cleavage of chymo- 
trypsin are apparently still held together by S-S- 
bridges. 

Considerable preliminary work has been de- 
voted to an investigation of the reaction with 
pepsin with cyanogen bromide (A. Arens). Un- 
like ribonuclease the stability and solubility of 
pepsin is unsuitable for reactions at acidic pH. At 
higher pH, such as pH 5 in acetate buffer, autolysis 
of pepsin is faster than the reaction with cyanogen 
bromide which reacts with the e-amino groups of 
lysine. In addition, pepsin denatures in a man- 
ner hitherto not properly recognized by forming 
4-5 definable fragments. 

The reaction of cyanogen bromide in the pH 
range of 4-8 is now under study with model amino 
acids, peptides and proteins (J. Schreiber). 

Probing the Tertiary Structure of Proteins by 
Selective Chemical Methods 

The controlled oxidative cleavage of ribonu- 
clease with limited amounts of N-bromosuccini- 
mide has led to a partial assignment of free and 



buried tyrosyl residues based on a quantitative 
assay of liberated amino terminal residues. This 
work has led to an extension in which positive bro- 
mine was transferred to ribonuclease from a high- 
molecular carrier. Eibonuclease in contact with 
such N-bromosuccinimide polymers lost its enzy- 
matic activity long before any modification of ty- 
rosine residues was noticeable by ultraviolet spec- 
trophotometry in situ (L. A. Cohen and J. G. 
Wilson). 

Three-dimensional structures of proteins at the 
present time are determined only by X-ray crystal- 
lography of crystals. There is no satisfactory 
method for the determination of 3-dimensional 
structures of proteins in solution under conditions 
of enzymatic activity. The use of chemically re- 
active polymers capable of selectively and rapidly 
modifying functional groups only on the surface 
may provide an important step forward in this 
problem. 

The principle of selective chemical modification 
of a protein with regard to its binding capacity 
has been utilized by N. M. Green in the laboratory 
of A. Neuberger (St. Mary's Hospital Medical 
School, London). He was able to adduce spectro- 
scopic evidence for the participation of trypto- 
phan residues in the binding of biotin by avidin. 
Dr. Green, who has joined the Laboratory of 
Chemistry, is extending these fruitful studies to 
the mechanism of binding involved in the complex 
formed between trypsin and trypsin inhibitor. 

A New Dimension in Analytical Sensitivity : Con- 
figurational Tautomerism of Cyclopeptides 

Thin layer chromatography of the cyclopeptide- 
antibiotic gramicidin A on silica gel has led to a 
resolution of gramicidin A into several discrete 
new tautomers whose concentration is dependent 
on the polarity of the initial solvent. This mobile 
equilibrium is interpreted in terms of configura- 
tional or conformational tautomers, possibly as a 
result of epimerization at the asymmetric carbon 
atoms involved as bridgeheads for the aminoetha- 
nol group which is assumed to span the cyclopep- 
tide ring by adding to two peptide carbonyls. 
These results suggest further application of the 
powerful tool of thin layer chromatography for 
the resolution of other open-chain or cyclic pep- 
tides into conformational tautomers. In the light 






146 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of these findings, the criteria for homogeneity of 
peptides or proteins may have to be redefined (S. 
Ishii). 

Rapid Configurational Analysis of Peptide Hy- 
drolysates by Combination of Enzymatic 
Assay With Quantitative Gas Chromatog- 
raphy 

Hydrolysates of gramicidin A were ti-eated sepa- 
rately with d- and L-amino acid oxidase. The 
residual amino acids were converted to the methyl 
esters of the dinitrophenyl derivatives and then 
assayed by quantitative gas chromatography. This 
rapid method clearly showed that gramicidin A 
consists of a valine-gramicidin (2 D-Val, 2 L-Val) 
and an isoleucine-gramicidin in which L-valine is 
partly substituted by L-Ileu. This novel technique 
is generally applicable to the study of peptides and 
proteins consisting of l- and D-amino acids (S. 
Ishii and R. Sarges) . 

Chemistry of Gramicidin A 

The complex splitting pattern of gramicidin A 
by N-bromoacetamide in aqueous ethanol contain- 
ing lithium acetate has been investigated by E. 
Gross and S. Ishii. A new cleavage method has 
been detected in the action of lithium aluminum 
hydride which liberated fragments containing 
lysine, valine and alanine as amino terminal resi- 
dues. Catalytic hydrogenation of gramicidin over 
rhodium on alumina in acetic acid containing per- 
chloric acid led to the uptake of 4 moles of hydro- 
gen with no change in the ultraviolet spectrum. 
The fragmentation of choice has been found in 
methanolysis at room temperature, a procedure 
which leaves the tryptophan residues fully intact. 

Stereospecific Synthesis of 4-Hydroxyproline 
and y-Hydroxyornithine 

In collaboration with Prof. N. Izumiya, head 
of the Dyst. Biochemistry, Kyushu University, 
the stereochemical investigation of the conversion 
of allylglycine to M/'<?o-y-hydroxyornithine and 
alio hydroxyproline has been completed. The in- 
termediate c^s-bromoaminolactone lends itself to 
the preparation of several other trifunctional 
amino acids such as the y-hydroxy-S-N-hydroxy- 
ornithine, the "prosthetic group" of ferrioxamines 
and ferrimycines from Actinomycetes. 



Synthesis and Stereochemistry of cis- and trans- 
Hydroxyproline, a New Amino Acid of 
Widespread Occurrence 

£nms-3-Hydroxyproline has been prepared in 
70% yield by stereoselective hydroboration of 3,4- 
dehydroproline. c?s-3-Hydroxyproline, prepared 
from this material via sodium borohydride reduc- 
tion of the 3-keto compound, was identical with 
the amino acid discovered in the hydrolysate of 
telomycin by F. Irreverre. K. Morita and F. Saki- 
yama have completed a one-step synthesis of 3- 
hydroxyproline by reacting aminomalonic acid 
with the addition product of acrolein to sodium 
bisulfite in a buffer solution. By modification of 
the conditions yields up to 50% have been obtained 
of mixtures of cis- and £nm.<?-3-hydroxyproline 
whose ratio depended on the nature of the buffer 
system and the cations present. 

Selectively tritiated 3-hydroxyproline has been 
made accessible by K. Morita to Prof. E. Katz, 
Dept. of Microbiology, Georgetown University, 
for incorporation studies into the peptide part of 
actinomycin by Streptomyces antibioticus. 

Congener of Actinomycin 

In collaboration with E. Katz and H. Weiss- 
bach, F. Marki has found evidence for the exist- 
ence of a congener of actinomycin in the culture 
medium of growing Streptomyces antibioticus. 
At least three different acidic compounds contain- 
ing the yellow phenoxazine chromophore and the 
same number of amino acids as actinomycin have 
been purified by differential extraction and chro- 
matography. These new congeners may differ 
from actinomycin by the opening of one or two of 
the lactone rings. Whether they may be pre- 
cursors or metabolites of actinomycin still remains 
to be seen. 

Studies on Dehydrobufotenine, the Major Ingre- 
dient of the Parotid Gland of Bufo marinus 

The synthesis of dehydrobufotenine and its nor 
and bisnor derivative has been started in coopera- 
tion with Prof. A. Burgstahler of the Univ. of 
Kansas and the Regis Chemical Co. under a Psy- 
chopharmacology contract. In addition to this 
ring synthetic approach starting from N-tosyl- 
l,2,3,4-tetrahydro-8-hydroxyquinoline, Dr. John 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



147 



Daly is investigating the direct conversion of sero- 
tonin to tricyclic analogs of dehydrobufotenine by 
selective oxidation with Fremy's salt. 

A number of indolevinylamines, previously 
thought to represent dehydrobufotenine, have 
been synthesized and characterized in cooperation 
with the Regis Chemical Co. These indolevinyl- 
amines are not sufficiently stable to permit their 
evaluation as psychopharmacological agents in 
studies with intact animals. 

The Venom of the Arrow Poison Frogs (Dendro- 
bates) of Southern Colombia 

F. Marki has participated in an expedition to 
the Choco Jungle region of southwestern Colombia 
and has succeeded, in cooperation with Explorer- 
Zoologist Mrs. Marte Latham, to collect for the 
first time a sizeable amount of Colombian arrow 
poison frogs. By skinning the frogs and extract- 
ing the skins immediately after collection he was 
able to prepare stable extracts of the venom which, 
even before purification, kills mice in a dosage of 
fractions of one gamma. Purification by column 
chromatography and countercurrent distribution 
has so far led to a 30-fold increase in the activity 
of the venom in 90% yield. It appears that the 
venom belongs to a chemical class hitherto not rep- 
resented in the animal kingdom. 

Specificity of Dopamine-/?-Hydroxylase 

Dr. Daly has made a careful investigation of the 
potent isosteric inhibitors of dopamine-/?-oxidase, 
e.g., derivatives of benzylhydrazine and O-benzyl- 
hydroxylamine. None of these inhibitors has been 
found to be a substrate for the enzyme. 

New Catecholamine Metabolites 

6-Hydroxydopamine and 6 -hydroxy - (nor) - 
epinephrine have been converted to their O-meth- 
ylation products enzymatically in vitro and in 
vivo. 2,5-dihydroxy - 4 - methoxyphenethylamine 
and -phenethanolamine are significant metabolites 
of dopamine and possibly also of norepinephrine 
(J.Daly). 

Chemistry of Adrenaline 

In collaboration with Dr. R. A. Heacock, Univ. 
Hospital, Saskatoon, Dr. J. Daly has investigated 
the NMR spectra of a large number of oxidation 
products of (nor) epinephrine, isoproterenol and 



3,4-dihydroxynorephedrine. The evaluation of 
these spectra has provided unambiguous proof for 
the position of the halogen in iodo and bromo- 
aminochromes, which is 7 and not 2, as has been 
assumed for the past 25 years. 

Metabolic N-Methylation of a Purine 

In collaboration with Dr. Julius Axelrod, Dr. 
J. Daly has established the first enzymatic methyl- 
ation of a normally-occurring purine. An enzyme 
occurring in rabbit lung converts adenine to 
3-methyladenine. Certain other purines are also 
methylated by this enzyme, but not pyrimidines. 
So far 3-methyladenine could not be detected in 
rabbit lung, either in the free form or bound in 
RNA or DNA. 

Oxidation Mechanisms of Tocopherol and Ubi- 
quinones and Their Metabolic Significance 

Drs. Diirckheimer and Cohen have found that 
the ionic oxidation of a-tocopherol leads, via a hy- 
droxydienone intermediate, to a-tocopheroqui- 
none. On the other hand, radical oxidation leads 
to dimeric products exclusively. Since autooxida- 
tion in tissues is probably a radical process, the 
failure to observe the quinone as a metabolic prod- 
uct of tocopherol oxidation is readily explained. 
However, dimeric materials, identical with those 
found by chemical methods, have recently been iso- 
lated with animal tissues. 

Studies on the oxidation of simple quinol phos- 
phates demonstrates that only 20% of the oxida- 
tive energy is conserved as metaphosphate, the re- 
mainder being lost by hydrolytic formation of in- 
organic phosphate. Such studies indicate oxida- 
tion of a ubiquinol phosphate to be the most likely 
pathway for synthesis of the desired dienone phos- 
phate. 

These chemical studies have a direct bearing on 
the fate of tocopherol in metabolism and in that 
respect may help to reveal its possible significance 
as an antioxidant or its role hi fertility and (ani- 
mal) muscular dystrophy. 

Dienone-Phenol Tautomerism and Its Signifi- 
cance in Metabolic Processes 

For some time Dr. Cohen and Mr. Jones have 
been studying the chemistry of phenols and the 
methods for increasing the contribution of die- 
nones to their structures. Such knowledge has a 



705-685—63- 



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148 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



direct bearing on tyrosine metabolism, thyroxine 
biosynthesis, aromatic hydroxylation and oxida- 
tive phosphorylation. 

From a study of the effect of substituents on the 
pK values of hindered phenols, it has been possi- 
ble to evaluate the importance of solvation at vari- 
ous sites on the phenolic ring. A method for cor- 
relating the ultraviolet spectra of phenols with 
the nature of para substituents has been achieved 
for the first time ; similar correlations have been 
obtained for both infrared and nuclear magnetic 
resonance spectra. From such data, it has been 
possible to predict and to demonstrate experimen- 
tally that certain phenols will react exclusively as 
dienones or as quinonemethines. 

Synthetic and Degradative Studies of Nucleic 
Acids 

G. W. Milne, John M. Steele and L. A. Cohen 
have synthesized dihydrothiophene and dihydro- 
thiopyran as potential blocking agents for the 2'- 
hydroxyl of ribotides. It has been shown that 
secondary hydroxyl groups react with the reagents 
and that the resulting thioacetals are cleaved eas- 
ily, in aqueous solution at 25°, by the action of 
silver or mercury ions ; the necessity for using acid 
or alkali for the removal of blocking groups is 
thus avoided. Adenosine monophosphate is con- 
verted to a fluorophosphate by the action of dini- 
trofluorobenzene and triethylamine in dimethyl- 
sulf oxide. The activated phosphate may then be 
converted to a thiophosphate by the action of hy- 
drogen sulfide. The utility of cyclic vinyl thio- 
ethers as blocking agents is further explored and 
methods are being developed for the selective ac- 
tivation of terminal phosphates in oligonucleotides 
with the aim of selective sequential degradation 
of nucleotides by chemical methods. 

Sea Cucumber Poison 

Holothurin, the poisonous saponin, isolated 
from certain sea cucumbers, has been purified and 
submitted to hydrolysis. The aglycone appears 
to be a C-27 or 28 steroid containing two methoxy 
groups, one double bond, and a five-membered 
lactone ring. During this work it was found that 
dimethyl sulfoxide acted as an excellent solvent 
for cleavage of steroidal digitonides. A nearly 
quantitative recovery of the sterols is possible. 
This has some advantages over the conventional 



method of cleaving steroidal digitonides (C. H. 
Issidorides, G. V. Nair, I. Kitagawa and E. 

Mosettig) . 

Sapogenins 

The structure of pennogenin, a steroidal sapo- 
genin, has been partially solved by degradative 
and nuclear magnetic resonance studies. The con- 
troversial position of the hydroxyl has been fixed 
at C-17 or C-20. In these studies it became of 
interest to synthesize 20-isocholestane, 17-isocho- 
lestane, and l7-iso,20-isocholestane. These com- 
pounds are of practical and theoretical significance 
in the steroid and triterpene field (G. V. Nair and 
E. Mosettig). 

Carcinogenicity and Structure 

A six-step synthesis of 3a,12a-dihydroxy-A 7 - 
cholenic acid from cholic acid has been perfected 
This precursor to apocholic acid, a known carcino- 
gen was desired for correlating carcinogenicity to 
structure (J. A. Waters and E. Mosettig) . 

Chemistry of Cortisone 

A number of sulfur analogs of corticoids and 
androgens have been synthesized for the CCNSC 
program. Among them, the enol acetate of 9a- 
methyl thioadrenosterone was found to exhibit 
high antigonadotropic activity. During the 
course of these preparations a novel rearrange- 
ment which consists in the rupture of the -C-N- 
bond at C-ll of the thiazoline ring and reattach 
ment to C-5 with formation of a thiazine ring was 
observed. This rearrangement occurred with 2' 
methoxythiazolino (4',lla ; 5',9a) -hydrocortisone 
and -dihydroadrenosterone (I. Kitagawa and E. 
Mosettig). 

Steroids of Insects 

In cooperation with the Entomology Eesearch 
Division of the Department of Agriculture, the 
major sterol present in the house fly was identified 
as campesterol. It was found that the fly selec- 
tively takes up the sterol from the CSMA media. 
It is of some interest to speculate on its relation- 
ship to cholesterol, a sterol required for insect 
growth. In conjunction with the above work, 
pure /^-sitosterol was needed for comparison pur- 
poses. It was prepared from stigmasterol by re- 
duction of 3,5-cyclostigmasterol (i-sterol) (J. A. 
Waters, J. A. Steele and E. Mosettig) . 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



149 



Steroid Biosynthesis in Plants 

In the work on the biogenesis of plant steroids 
(cooperating unit: U.S. Dept. of Agriculture) it 
was found that the leaves of D. spiculifora contain 
the sapogenin diosgenin, yamogenin and gentro- 
genin and the sterols ^-sitosterol and cholesterol. 
The presence of cholesterol in higher plants has 
been observed for the first time. It was further 
found that radioactive mevalonic acid was incor- 
porated into stigmasterol diosgenin and /3-sito- 
sterol in the leaves in the ratio 1 :2 :6. The higher 
specific activity of the steroids in the leaves as 
compared to that in the tuber suggests that the 
former is the active biosynthetic site and the latter 
a place of storage. The greater radioactivity of 
^-sitosterol as compared to stigmasterol indicates 
that stigmasterol is formed from /3-sitosterol by 
dehydrogenation contrary to animal steroid bio- 
synthesis. In solanum tuberosum, radioactive 
mevalonic acid appears to be preponderantly in- 
corporated into stigmasterol and /J-sitosterol. 
There is evidence that cholesterol is also present 
(R. D. Bennett, D. F. Johnson and E. Heftmann) . 

Microbial Hydroxylations of Steroid Alkaloids 

The steroidal alkaloids, solasodine and tomati- 
dine have been hydroxylated by the fungus Heli- 
costylum piriforme to yield products which have 
been unambiguously established. They are 9a- 
hydroxy-, lla-hydroxy- and 7/3-hydroxy-solaso- 
dine, and 7a-hydroxy-, 9a-hydroxy- and 7a,lla- 
dihydroxytomatidine. The steroidal sapogenin 
diosgenin has also been transformed microbiologi- 
cally into 7/3,1 la-dihydroxy- and lla-hydroxy-7- 
oxodiosgenin. A third component believed to be 
lla-hydroxy-7-oxotigogenin was also isolated. 
These are the first reported instances of the micro- 
biological transformation of steroidal alkaloids 
and sapogenins. They are not only useful in the 
synthesis of hormone analogs but also important 
in the understanding of steroidal metabolism in 
plants and animals ( Y. Sato, S. Hayakawa and A. 
Reine) . 

Role of Steroids in Cell Membranes 

Some doubts have been cast on Willmer's theory 
that steroid hormones regulate cellular perme- 
ability by specific interaction with lipids in cell 
membranes. In model experiments the steroid 
hormones failed to form stable monomolecular 



films and did not penetrate lipid monolayers (E. 
Heftmann) . 

Steviol as a Growth Factor in Plants 

Steviol whose structure was elucidated in this 
laboratory was found to produce significant 
growth response in the d-5 dwarf mutant of Zea 
mays. This demonstration of gibberellin activity 
outside of the group of natural gibberellins 
changes our present concept of the structural re- 
quirements necessary for gibberellin activity and 
also indicates a possible role of diterpenes in 
plants (cooperating unit: California Institute of 
Technology) (E. Mosettig). 

Steroid Nature of Florigen, the Flowering 
Hormone 

When inhibitors and radioactive precursors of 
steroid biosynthesis were applied to short day 
plants, floral induction was suppressed. Simul- 
taneously, the biosynthesis of stigmasterol and 
y3-sitosterol was inhibited. These experiments 
shed some light on the chemical nature of the flow- 
ering hormone (cooperating unit: California In- 
stitute of Technology). They may lead to meth- 
ods for the control of crop production and in the 
words of Prof. James Bonner, CALTECH, may 
have far-reaching effects on the world's food sup- 
ply (E. Heftmann). 

Qualitative Analysis of Steroids 

Thin-layer chromatography has been success- 
fully adapted to sterols, steroidal sapogenins and 
steroidal alkaloids as well as to cortocosteroids. 
This is the first practical method for resolving the 
C-25 epimers of sapogenins and the simplest 
method for the qualitative analysis of aldosterone 
yet devised. All adrenocortical hormones, in 
amounts of less than O.Oljug, can be separated and 
identified in 10 minutes. Useful correlations be- 
tween structural features and chromatographic 
mobility have been derived (R. D. Bennett and 
E. Heftmann) . 

Automatic Steroid Analyzer 

The performance of the steroid analyzer has 
been tested with respect to sensitivity, precision 
and accuracy. The efficiency of separation of 
steroids has been improved by a detailed study of 
elution gradients. Individual steroids and other 
migrants can be eluted at any desired interval by 






150 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



proper selection of gradient cams. The time previ- 
ously required for complete automatic quantitative 
analysis of complex mixtures containing all 7 
adrenocortical hormones has been reduced by one- 
half (D. F. Johnson, D. Francois and E. Heft- 
mann) . 

Infrared Fine Structure of Steroids 

A high-resolution infrared spectrophotometer is 
being used to obtain precise information for ab- 
sorption band studies and to provide spectra for 
Lorentzian analysis of absorption band envelopes. 
Analyses of this kind should provide the chemist 
with details of molecular structure which, until 
now, have been hidden in complex absorption en- 
velopes of the fingerprint region (H. K. Miller 
and Mrs. A. H. Wright) . 

Rotatory Dispersion of Sugar Lactones 

The Rudolph spectropolarimeter is being im- 
proved to provide constant schedules of half -inten- 
sity band width and precision thermostatting. 
The instrument has been used in a study of the 
ORD characteristics of D-ribonolactones. 

Microanalytical Services 

Approximately 8,200 analytical determinations 
were carried out for 130 of the NIH research staff 
and for several scientists in other Government 
agencies. These included about 7,900 routine 
microanalyses and approximately 275 nonroutine 
analysis requiring varying degrees of literature 
and laboratory investigations. 

LABORATORY OF BIOCHEMISTRY AND 
METABOLISM 

Carbohydrate Metabolism 

A. Reactions Involving Carbohydrate Polymers 
(Dr. J. Preiss and Dr. G. Ash well) 
Recent studies on the metabolism of polygalactu- 
ronic acid by cell free extracts of adapted bacteria 
have led to the isolation and identification of a new 
diketouronic acid, 3-deoxy-D-glycero-2,5-hexodiu- 
losonic acid. The initial monomeric reaction prod- 
uct, 4-deoxy-L-threo-5-hexoseulose uronic acid was 
found to be acted upon by a specific isomerase to 
produce the above diketo uronic acid intermediate. 
The latter, in turn, was reduced in the presence of a 
DPNH-linked dehydrogenase to form 2-keto-3- 
deoxy-D-gluconic acid. The subsequent metabolic 



pathway was shown to be identical to that previ- 
ously described for glucuronic, galacturonic, and 
alginic acid. 

(Drs. A. Ginsburg and B. M. Gesner) The in- 
triguing hypothesis that mammalian heteropoly- 
saccharides act as recognition surfaces for cellular 
interactions was investigated using the "homing" 
reaction of isologous P 32 -labeled lymphocytes. 
The "homing phenomenon" means that injected 
white cells congregate in certain sites in the animal 
body. It was found that the "homing phenome- 
non was destroyed by pretreatment with small 
amounts of glycosidic enzymes (crude clostridial 
extract) under conditions which had no effect 
upon the vital staining or motility properties of 
the lymphocytes. The effect of the glycosidic ac- 
tion was completely abolished by the presence of 
five sugars (L-fucose, D-mannose, D-galactose, 
N-acetylglucosamine, and N-acetylgalactosamine) . 
Strikingly, equal amounts of D-glucose exhibited 
no protective effect. The experiments suggest a 
hitherto unsuspected function of mammalian poly- 
saccharides. 

B. Reactions Involving Small Carbohydrate 
Molecules and Carbohydrate-Containing Co- 
enzymes (Dr. J. Preiss) 

A new nucleotide sugar, GDP-mannuronic acid, 
has been identified and shown to arise by the ac- 
tion of a specific pyridine-linked dehydrogenase 
upon GSP-mannose. The enzyme was partially 
purified from a cell- free extract of a bacterium 
which forms a mannuronic acid containing exo- 
polysaccharide. 

(Dr. P. J. O'Brien) The biosynthetic pathways 
for several unusual sugars which are present in 
the surface antigenic lipopolysaccharides of cer- 
tain microorganisms are under investigation. The 
novel sugar nucleotide, guanosine diphosphate-D- 
glycero-D-mannoheptose (GDPH) has been iso- 
lated from yeast and characterized. An enzyme, 
GDPH pyrophosphorylase, derived from yeast, 
cleaves this nucleotide to GTP and heptose-1-P. 
The reverse reaction apparently represents an 
activation step in the pathway of heptose incorpo- 
ration into polysaccharides. A similar study of 
D-f ucosamine biosynthesis is also underway. 

(Dr. A. Grollman) The biosynthesis of the 
oligosaccharides found in milk, both in vivo and 
in vitro, was studied. In vivo labeling experi- 
ments indicated that these oligosaccharides are 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



151 



built by stepwise addition of various sugars to 
D-glucose. In vitro, particle preparations from 
lactating mammary glands were found to catalyze 
the transfer of L-fucose from GDP-L-fucose to 
lactose, forming the naturally occurring trisac- 
charide, fucosyl lactose. The total synthesis of 
this trisacchardide from D-glucose can now be 
Avritten using known reactions. 

(Dr. F. Eisenberg, Jr.) Sucrose is the prototype 
of a homologous class of non-reducing plant oligo- 
saccharides which serve as storage nutrient for 
plants and fuel for animal organisms. The inter- 
glycoside linkage in these sugars has been shown 
in studies with transferases to be a high energy 
linkage, but it has not been possible, in these en- 
zyme studies, to differentiate the glucose-0 bond 
from the fructose- O bond with respect to their 
stability to cleavage. From consideration of the 
structure of groups surrounding the linkage, one 
would predict that the fructose-0 bond is the 
more labile. The results of experiments in which 
sucrose was hydrolyzed in H 2 18 with IR-120 
(H) cation exchange resin provide direct proof 
of this supposition. 

(Drs. Y. J. Topper, L. Laster, and S. Segal) 
It was previously reported that two subjects who 
had typical symptoms of congenital galactosemia 
in infancy and who now have no detectable P-gal 
uridyl transferase enzyme in their red cells are 
near normal in the capacity to oxidize galactose 
in vivo. Five tissues from one of these subjects 
have been examined in vitro for their ability to 
convert galactose-1-C 14 to C 14 2 . Of these, only 
liver compared favorably with tissue from control 
subjects. The finding of such phenotypic differ- 
ences among several tissues from a single galacto- 
semic patient indicates that galactosemia is a more 
complex disease entity than has been appreciated 
hitherto. 

Dr. M. Stetten) There exists a mammalian par- 
ticulate inorganic pyrophosphatase, which has 
been found to have transphosphorylation activity. 
It phosphorylates glucose on carbon-6 in the pres- 
ence of PP. The results of kinetic studies are 
compatible with the assumption of a single protein 
on the surface of which glucose and water compete 
for pyrophosphate. Glucose, rather than water, 
seems to be the preferred substrate. The possibil- 
ity exists that under suitable conditions this en- 
zymatic reaction may afford an alternative route 
for the formation of glucose-6-phosphate. 



Other Studies on Biosynthesis 

A. Thiamine (Dr. I. G. Leder 

The formation of thiamine phosphate is cata- 
lyzed by a bakers' yeast enzyme discovered in this 
laboratory, thiamine-phosphate pyrophosphoryl- 
ase. This enzyme promotes the condensation of 
the two ring moieties according to the following 
equation : 

"pyrimidine"-PP + "thiazole"-P 

Mg^ 
?=>thiamine-P + PP 

The enzyme has been purified 1,200-fold and 
crystallized. It sediments as a single peak in the 
ultracentrifuge (sedimentation constant approxi- 
mately 11) and moves as a single enzymatically 
active protein band on disc electrophoresis. 
Studies on mechanism indicate a direct reaction 
of the pyrimidyl and thiazyl esters without the 
participation of an activated pyrimidylenzyme 
intermediate. 

The enzyme "thiazole" kinase, which catalyzes 
the phosphorylation of "thiazole" to "thiazole" 
monophosphate, has been partially purified. 

B. Fatty Acid Synthesis (Drs. B. Bloom and 

D. W. Foster) 

As an extension of studies into the metabolic 
fates of the several classes of hydrogen, the in- 
corporation of tritium, derived from tritium oxide, 
into fatty acids and glucose by surviving tissues 
has been inactivated. Isotopic palmitic and stearic 
acids synthesized by rat liver slices in the presence 
of T 2 have been sequentially degraded in order 
to determine the intramolecular location of the 
tritium. It was found that the beta and delta 
positions contained more isotope than the alpha 
and gamma positions. This was unexpected since 
the hydrogens at beta and gamma are derived, as 
determined with soluble enzyme techniques, pri- 
marily from TPNH rather than from protons 
of the medium. These observations suggest that 
an exchange reaction, probably mediated by a 
flavoprotein, is involved in the system which 
utilizes TPNH for the reduction of carbon-carbon 
double bonds of fatty acids. Glucose synthesized 
by the same liver slices was found to contain 2-3 
times as much tritium at position 1 as at position 6. 
Whether this is due to the phosphomannose 
isomerase catalyzed reaction, reactions of the non- 



152 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



oxidative sequence of the hexose monophosphate 
shunt, or some other cause will be determined. 

Regulatory Mechanisms and Hormones 

A. (Drs. J. Cohen, A. R. Brenneman and Y. J. 
Topper) In continuation of studies on the meta- 
bolic effects of oxytocin and acetylcholine on rat 
mammary gland in vitro it has been found that 
the stimulation of glucose oxidation effected by 
these hormones is depressed by puromycin. The 
depression of the effects is a fimction of the de- 
gree to which puromycin inhibits protein syn- 
thesis. Subsequently it was found that whereas 
the hormones do not alter the incorporation of 
leucine-1-C 14 into total protein, they do appear 
to change the distribution of newly synthesized 
radioactive protein among the various cell con- 
stituents. Oxytocin has no such effect on the syn- 
thesis of protein by liver in vitro, nor does insulin 
have this effect on surviving mammary gland. 
The relationship between the effect on protein syn- 
thesis and the stimulation of glucose oxidation is 
under investigation. 

B. (Drs. F. Tietze, H. M. Katzen and D. Stet- 
ten, Jr.) Both immunological and adipose tissue 
assays indicate that the oxidation of reduced in- 
sulin in the presence of oxidized glutathione and 
the enzyme glutathione-insulm transhydrogenase 
leads to the formation of insulin-like activity in 
excess of that formed in the absence of the enzyme. 
This observation constitutes the first indication 
that an enzyme can influence the establishment 
of the "correct" disulfide linkages of a protein. 
It suggests that enzymes might be involved in the 
in vivo establishment of the three-dimensional 
structure of proteins subsequent to the formation 
of the polypeptide backbone. 

Nucleic Acids and Other Polynucleotides (Dr. 
M. F. Singer) 

Several new investigations on polynucleotide 
phosphorylase were completed. In one, the ar- 
senate-dependent breakdown of nucleoside di- 
phosphates by polynucleotide phosphorylase was 
found to be stimulated 10 to 100-fold by the addi- 
tion of oligonucleotides having unesterified C-3'- 
hydroxyl groups. The oligonucleotide acts cata- 
lytically and kinetic evidence indicates that the 
oligonucleotide acts as a second substrate in the re- 



action. A general mechanism involving the for- 
mation of a new phosphodiester bond between the 
nucleoside diphosphate and oligonucleotide and 
its subsequent arsenolysis was developed for this 
reaction. This mechanism also applies to other 
reactions catalyzed by the enzyme. 

(Drs. M. F. Singer and F. N. Brenneman) 
Polymerization experiments were carried out with 
various ratios of GDP to the other nucleoside di- 
phosphates. Under certain conditions there is an 
enrichment of guanylic acid residues in the poly- 
mer, along with an inhibition of reaction rate. 

(Drs. M. F. Singer, O. W. Jones and M. N. 
Nirenberg) It has also been found that the effi- 
ciency of a synthetic copolymer containing gu- 
anylic acid, poly GU, when acting as messenger 
RNA, is related to the extent of secondary struc- 
ture in the polymer. The data strongly suggest 
that messenger RNA must be single stranded and 
without extensive intramolecular hydrogen bond- 
ing. 

( Dr. L. A. Heppel) A study of specific polymer 
inhibition of polynucleotide phosphorylase has 
shown that the enzyme is specifically inhibited if 
nucleoside diphosphate and polymer have bases 
that form a hydrogen-bonding pair. For example, 
poly A inhibits UDP polymerization and poly I 
inhibits ADP polymerization. In the latter case 
it is possible to show that the enzyme inhibition is 
sharply temperature-sensitive, as if a complex 
were being "melted out." 

(Dr. M. N. Lipset) As mentioned above, gu- 
anylic residues in polynucleotides appear to have 
special properties. Guanylic polyribonucleotides, 
even as small as GpG, have a strong tendency to 
aggregate, forming a viscous, high-molecular 
weight complex that is almost completely inacces- 
sible to enzymic attack. This process of aggrega- 
tion makes it impossible to separate guanine oli- 
gonucleotides by ordinary methods. However, it 
was discovered that they can be separated by 
DEAE chromatography in the presence of 7 M 
urea. The pure guanine oligonucleotides, thus 
made available, are now being investigated as to 
their own interactions as well as complex forma- 
tion with cytosine-containing polynucleotides. 

(Drs. M. Gellert, M. N. Lipsett, and D. R. Da- 
vies) The monomer, 5-GMP, is able by itself to 
form a viscous aggregate, of highly ordered struc- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



153 



ture. These special properties of guanylic resi- 
dues are probably of great significance in nucleic 
acid function. 

(Drs. D. R. Harkness and R. J. Hilmoe) Alka- 
line phosphatase from E. coli has now been highly 
purified and freed of nuclease and diesterase ac- 
tivity. It was possible to use this enzyme as a 
reagent to remove specifically the 5'-terminal 
phosphate from s-RNA. It was found that re- 
moval of the terminal phosphate did not injure 
the ability of s-RNA to accept amino acid or to 
transfer amino acid to the ribosome. 

Biochemical Studies of Lysogeny (Drs. D. Korn 
and A. Weissbach) 

Biochemical studies of lysogeny in E. coli K12 
have shown that the DNA polymerase present in 
these cells is the same as that found in lysogeni- 
cally induced cells. However, lysogenic induction 
of E. coli K12 A has been shown to cause the forma- 
tion of a specific new deoxyribonuclease whose 
properties are being studied. This enzyme is also 
found after infection of K12S cells with lethal 
mutants of the phage A. It has also been found 
that lysongenic K12 cells which require thymine 
(K12 A Thy) can be induced to form mature A 
if they are suspended in a thymineless media for 
a certain time and then are resuspended in com- 
plete media. 

Enzymatic Utilization of Model Compounds (Dr. 
W. B. Jakoby) 

Continuing studies on the enzymatic utilization 
of model compounds have led to the isolation of 
three enzymes catalyzing deamidation, from three 
separate microorganisms. The enzymes differ 
from each other in their substrate specificity re- 
acting with acetamide, butyramide, and benzam- 
ide, respectively. Preliminary experiments indi- 
cate the possibility of a transfer reaction with 
hydroxylamine, resulting in the formation of the 
appropriate hydroxamic acid, as well as a de- 
amidation of an amide with the f ormation of the 
respective free acid. 

(Dr. A. J. Aspen) Studies on the oxidation of 
threonic acid have resulted in the isolation of a 
highly purified preparation from a pseudomonad 
which catalyzes the DPN-linked oxidation of thre- 
onic acid to /3-keto threonic acid. The latter, in 
turn, is decarboxylated to form dihydroxy acetone. 



LABORATORY OF EXPERIMENTAL 
PATHOLOGY 

Anatomical Pathology 

This Laboratory continues to provide consulta- 
tive pathology services to the Division of Indian 
Health and other facilities of the Public Health 
Service which has stimulated interest in aspects of 
geographic and environmental pathology. In ad- 
dition, the specimens provide a source of human 
material for histochemical and other investiga- 
tions. There has been a substantial increase par- 
ticularly in the number of surgical specimens sub- 
mitted. Problems related to sarcoidosis, diabetes, 
dietary hemosiderosis, cholecystitis, and to athero- 
sclerosis and its complications are of particular 
interest. Consultative service to two Korean 
Charity Hospitals has resulted in the collection of 
more than 50 cases of fatal Pneumocystis carinii 
infection in children. 

In addition to the research projects summarized 
separately, certain scientists in other laboratories 
received advice from members of our staff, partic- 
ularly in pathologic anatomy. Our histopatho- 
logic preparation unit also took part in this coop- 
erative effort by cutting and staining sections from 
about 1,700 animals this year for 18 investigators 
not in laboratories of NIAMD. 

Pathologic studies of tissues from nutritional 
experiments under the direction of Dr. F. Daft, 
NIAMD, are continuing. Subjects currently 
under investigation include acute hemorrhagic 
renal necrosis in choline- deficient rats and dietary 
hepatic necrosis in vitamin E and selenium de- 
ficient rats (Dr. Horn). 

Altitude Studies 

The effects of age and exercise on altitude toler- 
ance in rats continue to be investigated in coopera- 
tion with Dr. Altland. It was found that young 
adult rats withstood exposure to 34,000 feet better 
than younger or older animals and that exercise 
during exposure reduced tolerance by 3^,000 feet. 
Females fared better than males except in late 
pregnancy. The poor altitude tolerance of older 
rats did not seem to be related to myocardial de- 
generation, chronic nephrosis, or chronic murine 
pneumonia of moderate severity. More severe 
lung lesions, characterized by the presence of mul- 
tiple large bronchiectatic cavities containing 



154 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



necrotic exudate, had a pronounced effect in reduc- 
ing survival to altitude exposure and were partic- 
ularly frequent in older males (Dr. Higlnnan) . 

Bacterial Endocarditis 

Evidence was obtained that the marked suscep- 
tibility of high altitude rats to bacterial endocar- 
ditis is not due merely to hyperactivity of the 
adrenal or to impaired antibody formation. It re- 
quired the presence of both polycythemia (in- 
creased cardiac work load) and hypoxia (addi- 
tional deleterious factor). It is postulated that 
hypoxia causes biochemical and enzymatic changes 
that impairs the ability of host phagocytic cells 
to destroy certain bacteria. A similar explanation 
is proposed to account for the development of bac- 
terial endocarditis in 82-100% of rats maintained 
in a cold room at 1.7° C for 1 to 36 days before and 
7 days after an intravenous injection of a culture 
of Streptococcus mitis, JH 26. In control rats 
maintained in a normal temperature environment 
and in a group of cold rats returned to a normal 
temperature environment after the bacterial inocu- 
lation, the incidence of induced severe bacterial en- 
docarditis was only 16-19%. In rats acclimatized 
to cold 35-36 days, the mortality rate was lower 
and the length of survival was longer than in rats 
placed in the cold room only one day before the 
bacterial inoculation (Dr. Highman). 

The tissue source of the serum enzymes which in- 
crease in animals subjected to various stress is 
being investigated. Electrophoretic separation of 
the elevated serum enzymes into their isozymic 
components will be utilized to elucidate this prob- 
lem (Dr. Garbus). 

Cytogenetic Studies 

(1) In studies of the karyotypes in patients 
with congenital disorders, the following abnormal 
karyotypes have been found among 50 patients re- 
ferred by clinical investigators of NHI, NINDB, 
NCI and private physicians : 

(a) Down's Syndrome with 47/ trisomy 21 (6 
cases) 

(b) Mental retardation (?) with 47/trisomy 
18 (1 case) 

(c) Phenotypic female with Turner's syn- 
drome with 45/XO (4 cases) 

(d) Phenotypic female with Turner's syn- 
drome with 45/XO and 46/mosaicism (1 
case) 



(e) Identical twin sisters of wnich one is a 
typical Turner's and the other was appar- 
ently normal and menstruating. How- 
ever, both had the same chromosomal ab- 
normality, i.e. mosaic 45/XO and 
47/XXX 

(f) Female with 47/XXX (1 case) 

(g) Phenotypic male baby with androgenital 
syndrome with 46/XX (1 case) 

(h) Female with testicular feminization syn- 
drome 46/XY (1 case) 

(i) Male with Klinefelter's Syndrome 47/ 
XX Y (3 cases) 

(j) Male with 47/XYY (1 case) 

(k) True hermaphrodite with 46/XY (1 
case) 

(2) In cooperation with DBS-LVE chromo- 
some studies have been carried out on an SV 40 
virus susceptible cell line designated BS-C-1 es- 
tablished from kidney tissue of Oercopithecus 
aethiops as part of their program on the develop- 
ment of tissue culture lines for use in testing of 
vaccines. This line is now in its 125th passage and 
has retained its sensitivity to infection with SV 
40 virus although the chromosome complement 
was found to have changed from diploid (up to 
the 30th passage) to subdiploid (at the 41st pas- 
sage) and finally to subtetraploid at the 111th 
passage. 

(3) In cooperation with the NCI-MB, studies 
of the chromosomes of malignant cells in patients 
with leukemia and related diseases have continued. 

(a) Studies on the chromosomes of a patient 
with lymphoblastic lymphosarcoma, leukocytosis, 
eosinophilia and granulocytic hyperplasia of the 
bone marrow revealed a consistent chromosomal 
abnormality in the bone marrow specimens taken 
at various periods, before and after treatment and 
also in one lymphnode aspirate. There were con- 
sistently 2 chromosomes missing, one each in group 
6-12 and 13-15, respectively. Instead, two new 
chromosome types were present. One was very 
similar to pair 3 and the other was acrocentric and 
slightly longer than pair 21. The skin fibroblast 
and peripheral leukocytic cultures had a normal 
female karyotype, thus excluding the possibility 
that the patient had an abnormal chromosome 
constitution. 

(b) Of 40 patients with chronic myelogenous 
leukemia studied, 34 revealed the so-called Ph 1 
chromosome (a small chromosome replacing one 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



155 



of group 21-22 in the marrow cells). The cells Carcinogenicity of Cycas circinalis L. 



with this specific marker persisted after therapeu- 
tic treatment and also during remission. 

Of 32 treated patients with chronic myelogen- 
ous leukemia, 9 had improvement in their bone 
narrow to the extent that erythroid elements com- 
posed 20-60% of the nucleated cells. In 6 of these 
9 patients, 100% of the scored metaphases in the 
marrow contained the Ph 1 chromosome. Differen- 
tial counts of cells in mitosis on Giemsa stained 
marrow smears from these 6 patients showed 30- 
80% of the mitoses to be of the erythroid series. 
Thus it may be assumed that the erythroid as well 
as the myeloid cells of the marrow from CML pa- 
tients apparently contain the Ph 1 chromosome. 

(c) Acute Leukemia. "We have studied the 
chromosomes in bone marrow from 31 ALL and 
21 AML patients. The majority of the patients 
with acute leukemia have a diploid modality with 
a small range of aneuploid cells. There is no spe- 
cific pattern in the few ehromosomally abnormal 
cases. They could be irregularities to be expected 
during progression of the malignant disease. But 
if chromosomal abnormalities were an expression 
of the degree of malignancy, the incidence and 
variety of such abnormalities might be expected 
to correlate roughly with the acuteness of the leu- 
kemia. However, from the information available 
it does not appear that the abnormal cases were 
more acute, either clinically or hematologically, 
than those found to be of normal karyotype, ex- 
cept perhaps for some so-called terminal cases in 
the blast stage where we found numerical and 
structural changes in 4 out of 5 cases examined. 
The relation between cancerogenesis and chromo- 
somal changes in some of our cases is alternatively 
(1) autonomy induced by the chromosome 
changes, either by these changes alone or by the 
combined effect of the changes and factors agents 
such as virus or (2) autonomy induced prior to 
chromosome change. Since about 75% of the 
acute leukemias studied by us have a dipoid stem- 
line with apparently normal karyotype, we believe 
that these apparently normal cells were malignant 
and that the second alternative seems to be more 
likely for our acute leukemia cases. In acute leu- 
kemia evidently the karyotype may remain normal 
after the change into malignancy (Dr. Tjio). 



Interst in the nut from Cycas circinalis Z., origi- 
nated when a search for a possible neurotoxin was 
undertaken which might, in part at least, contrib- 
ute to the unusually high incidence of amyotrophic 
lateral sclerosis on the island of Guam (Drs. Kur- 
land and Whiting of NINDB). Experiments 
were set up in collaboration with Dr. Mickelsen of 
NIAMD to investigate the effect on the central 
nervous system of rats of the untreated dried, 
powdered nut. Although no histological evidence 
of a neurotoxin in the nut was found, benign and 
malignant metastasizing neoplasms, epithelial and 
mesenchymal in type, were found singly or in 
various combinations in livers, kidneys and occa- 
sionally in the lung in rats which had been on one 
of the experimental diets for more than three and 
one-half months. The absence of similar pro- 
liferative and neoplastic lesions in appropriate 
control groups strongly indicated the presence of a 
carcinogenic factor in the untreated nut. The neo- 
plastic and hepatotoxic effects are similar to those 
produced by nitrosamines. Investigations are 
being continued to establish the nature of the car- 
cinogenic agent (Dr. Laqueur). 

Enzyme Histochemistry 

Work has continued on the characterization of 
the mammalian peptidase specific for the hydroly- 
sis of N-terminal a-1-glutamyl and aspartyl resi- 
dues. This enzyme has been partially purified and 
characterized by biochemical techniques in con- 
junction with Dr. J. E. Folk of NIDR. It is cal- 
cium activated and EDTA (versene) inhibited, 
and is demonstrable histochemically in such tissue 
sites as guinea pig pancreatic duct epithelium and 
islets of Langerhans and in human and rat glo- 
merular epithelium and proximal tubule brush 
borders. No hydrolysis of N a -substituted glu- 
tamyl peptides or of the d-enantiomorph was ob- 
served in the presence of high concentrations of the 
enzyme. The above data and the absence of hy- 
drolysis of N(a-d-glutamyl) / 8-naphthylamide and 
N (Ni a -benzoyl-a-d-glutamyl ) /?-naphthylamide in 
both the histochemical and biochemical systems 
and of N-carbobenzoxy-a-1-glutamyl-l-phenylal- 
anine in the biochemical system indicates that the 
enzyme is stereospecific, exhibits primarily exo- 



156 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



peptidase activity and has a relative specificity 
for the hydrolysis of N-terminal 1-dicarboxylic 
acid peptides (Drs. McMillan and Glenner). 

In order to relate histochemical enzyme charac- 
teristics with those of a biochemical system, a 
technique has been devised for determining en- 
zyme kinetic constants in a histochemical system. 
By means of this technique which utilizes a con- 
stant chromatic endpoint, a double reciprocal plot 
of velocity and substrate concentration (Line- 
weaver-Burk) can be constructed for the activity 
of an enzyme in a specific tissue site. From these 
data and using competitive inhibitors the kinetic 
constants K m , V m and K ; can be determined. In 
this way it has been possible to show the presence 
in human mast cells of an enzyme having both 
esterase and amidase activity and characteristics 
similar but not identical to trypsin. This tech- 
nique is applicable to instrumental measurement 
and such an application is in progrses (Drs. 
Hopsu, McMillan and Glenner) . 

Eosinophilic Meningo-encephalitis 

In recent collaborative studies with Drs. Rosen 
and Weinstein of NIAID an eosinophilic menin- 
goencephalitis in man presumably has resulted 
from cerebral involvement by a metastrongylid 
lung worm of rats, Angiostrongylus cantonensis. 
Pathologic studies of monkey brain and spinal 
cords at various intervals after oral ingestion of A. 
cantonensis larvae demonstrated severe and dif- 
fuse eosinophilic meningo-encephalitis and para- 
sites in the brain at day 17 after infection. The 
lesions in the experimentally infected monkeys 
closely resembled those seen previously in the 
spontaneous human infection and differed greatly 
from lesions produced in the rat, which is the nat- 
ural host (Dr. Laqueur) . 

Experimental Obesity 

The pathogenesis of the obesity in mice which 
results from the administration of goldthioglucose 
was restudied in cooperation with Drs. Schwartz 
and Cronkite of the Brookhaven National Labora- 
tory. Using activation analysis and radioautog- 
raphy as well as serial sections and routine 
histologic methods, it could be shown that gold- 
thioglucose damages several areas in the brain, 
including the hypothalamic area which contains 
the "satiety" center. The satiety center is often 
damaged only marginally by goldthioglucose. 



Only those animals in which this center was en- 
tirely destroyed developed obesity (Dr. Brecher). 

Hematology 

The concept proposed earlier that short lived 
macrocytes are produced after intense erythroid 
stimulation received further support with the 
demonstration of their production in iron deficient 
animals or human beings in response to iron 
therapy (Drs. Moo res, Stohlman and Brecher) 
and in the regenerative phase of post irradiation 
anemia (Dr. Stohlman). Splenectomy did not 
affect the life span of these cells (Drs. Moores, 
Stohlman). 

Further studies on the growth of small periph- 
eral blood lymphocytes demonstrated that RNA 
synthesis begins about 24 hours prior to DNA 
synthesis ; the morphologic changes in culture were 
further delineated by electron microscopy (Drs. 
Epstein, Stohlman, Brecher, Tanaka). 

Proliferative capacities of peripheral blood cells 
are being studied by autoradiographic techniques 
which measure m vitro incorporation of tritiated 
thymidine into DNA. Blood withdrawn from 
patients with infectious mononucleosis during the 
first 2-3 weeks of illness has a marked increase 
in the percent of mononuclear cells in DNA syn- 
thesis as compared with normal controls. The per- 
centage decreases steadily as the illness progresses. 
In contrast, the percentage of mononuclear cells 
in DNA synthesis in patients with leukemia varies 
widely and in many instances is normal. This 
would imply that high proliferative capacity is 
not a requisite for malignancy (Drs. Epstein and 
Brecher) . 

Hemoglobin 

A sub-molecular mechanism of sickle cell forma- 
tion was deduced from the optical rotary disper- 
sion data and a series of experiments with a scale 
model building of the N-terminal segment of the 
/3-chain where the genetic abnormality of sickle 
cell hemoglobin (HgbS) is located. This defect 
consisting of the substitution of valine for the 
glutamic acid present in normal hemoglobin 
(HbA) at position 6 permits the stabilization of 
the N-terminal polypeptide chain of HgbS in a 
ring structure. The amino terminal Valyl residue 
and the abnormal Valyl of Hb-S molecule can in- 
terlock by van der "Walls forces of hydrophobic 
bonds ; this allows a ring structure to form from 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



157 



the N of Histidyl to the carbonyl group of 
Threonyl residue. This conformation appears to 
allow stacking of the molecule which results in a 
tactoid (or sickle cell) formation. 

When the 6th residue is glutamyl as in Hb-A, 
it appears that this ring closure does not occur 
due to electrostatic repulsion of the carboxyl 
groups. In Hb-S at 38° this segment of the pep- 
tide chain is stabilized through this cyclization 
by hydrogen bonding, thus the freedom of rotation 
about bonds in rings formed is restricted. This 
is the condition necessary and sufficient for one of 
the Kautzmann and Eyring rules which states: 
"Those influences which tend to restrict freedom of 
orientation about bonds will tend to increase the 
order of magnitude of the optical rotation." But 
at 0° the hydrogen bonds under question appear 
to be transferred in a process of hydration (be- 
cause hydrophobic bonds are weaker at 0°) and 
the chain appears to assume a conformation of no 
ring formation as in the /3-chain of Hb-A; thus, 
the numerical value of the optical rotation should 
therefore be diminished at 0° for Hb-S hemoly- 
sate. 

The optical rotatory dispersion studies of the 
Hb-A and Hb-S hemolysates showed that the am- 
plitude of the positive Cotton effect is increased 
reversibly by about four times when the sickle cell 
hemolysate was warmed to 38° from 0° ; the nor- 
mal hemolysate does not have this property. An- 
other physical property of Hgb-S that may be 
explainable in terms of the conformational altera- 
tions of the molecular model outlined above is the 
negative temperature coefficient of gelation, i.e., 
Hb-S hemolysate gels upon deoxygenation at 38° ; 
this gel melts at 0°. 

The mercapto-mercapto interactions in the nor- 
mal hemoglobin have been compared with those 
in the sickle cell hemoglobin ; there is no signifi- 
cant difference in the mercapto-mercapto inter- 
action constants between these hemoglobin mole- 
cules. 

The mercapto-mercapto interaction constants 
between the mercuric ion, methyl mercuric hy- 
droxide, and ethyl mercuric hydroxide have been 
evaluated using the normal human and the horse 
hemoglobins. The energy barrier due to steric 
hindrance for the methyl group amounts to about 
2 kilocalories mole" 1 . This finding is consistent 
with the basic notion of the steric hindrance 
theory postulated for the concept of the mercapto- 



mercapto interactions. (This is analogous to the 
well known heme-heme interactions in the process 
of 2 binding ; when mercurials are in the process 
of binding the mercapto groups, the mercapto- 
mercapto interactions take place in the molecule) 
(Dr. Murayama). 

Histochemistry of Mucopolysaccharides 

Interest in differentiating and characterizing 
mammalian mucopolysaccharides by histochemical 
methods has continued. Efforts have been directed 
primarily toward development of specific histo- 
chemical methods for localizing the different muco- 
polysaccharides, secondarily toward application of 
these methods. Modification of a previously de- 
veloped diamine method to include a mixture of 
diamines and ferric iron has provided a technique 
which is sensitive and almost completely specific 
for acid mucopolysaccharides and by several varia- 
tions in the teclmique differentiates between sul- 
fated and non-sulfated (usually sialic acid contain- 
ing) entities. With or without added iron, the 
mixed diamines reveal further a number of as yet 
unexplained difference between members in both 
categories. It has also been found that an alcian 
blue-safranin sequence, in which the pH of the 
alcian blue step is varied, likewise distinguishes 
sulfo- from sialo- mucins and with alcian blue at 
pH 0.5 distinguishes further between types of sul- 
fomucins. The mixed diamine procedure differ- 
entiates either sulfated or sialic acid containing 
mucins which possess vie glycols (and hence pre- 
sumably hexose) in proximity of the acid group. 
Mucins losing the diamine staining as a result of 
prior oxidation with periodate are those identified 
as having vie glycols in stearic proximity to the 
acid groups. This property although not a recog- 
nized characteristic of any biochemically known 
mammalian acid mucopolysaccharide can be 
demonstrated by the histochemical procedure. It 
can also be clearly shown with a sequence of di- 
amine followed by alcian blue or the Hale colloidal 
iron stain for acid mucopolysaccharides. In this 
instance the periodate reactive (hexose containing) 
acid mucosubstances lose alcian blue or colloidal 
iron staining after a periodate diamine step in 
contrast with the periodate unreactive polymers 
which retain such staining unimpaired by oxida- 
tion (Dr. Spicer). Application of these methods 
has been undertaken in attempting to characterize 
mucosubstances in pathologic states including 






158 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



autopsied cases of cystic fibrosis, malignancies of 
various sites and certain inflammatory lesions 
(Drs. Spicer and Duvenci). 

Hypersensitivity 

In a previous study of alterations in circulating 
leukocytes during induction of the generalized 
Shwartzman reaction in rabbits, prominent meta- 
chromatic cytoplasmic granules were noted in cir- 
culating neutrophiles. Histochemical and auto- 
radiographic studies have indicated that these 
metachromatic granules are similar to and presum- 
ably derived from the azurophile granules of the 
immature granulocytes of the bone marrow. The 
metachromatic substance of the azurophile gran- 
ules, previously assumed to be ribonucleic acid, has 
been characterized as a sulfate-containing acid 
mucopolysaccharide, in combination with a strong- 
ly basic protein. A similar constituent, occurring 
in certain granules in immature eosinophiles, is less 
basophilic, and several observations indicate that 
strong bonding between acid groups of the poly- 
saccharide and basic groups of the protein may 
account for its weaker affinity for basic dyes (Drs. 
Horn and Spicer). 

Immunochemical Studies 

A. Glyceraldehyde-S-phosphate dehydrogenase 

Previous investigations dealing with the local- 
ization of glyceraldehyde-3-phosphate dehydro- 
genase (GAPD) by fluorescent antibody technique 
and its distribution in adult striated skeletal 
muscle of the cockroach have been extended to 
include a study of the presence and distribution of 
the enzyme in the developing myoblasts. The en- 
zyme is found in higher concentration in the heart 
myoblast than in the skeletal myoblast and is local- 
ized in particular but not all mitochondria. In 
the culture of the heart myoblast the enzyme has 
been found in highest concentration in the small 
fibrocytes which adhere closely to the myoblast 
(Dr. Emmart in collaboration with Dr. Kominz 
of NIAMD and Dr. J. Miquel formerly of 
NINDB). 

The localization of this enzyme (GAPD) has 
been further studied in rat kidneys. GAPD was 
not observed in the glomeruli and varied in con- 
centration in different areas of the tubules. It 
was especially noted in adventitial cells of the 



medulla (Drs. Emmart and Spicer in collaboration 
with Dr. Schimke, NIAMD) . 

B. Prolactin 

Immunochemical studies have been carried out 
in collaboration with Drs. Condliffe and Bates 
with fractions of prolactin prepared by chroma- 
tography on DEAE column and also by counter- 
current distribution (Dr. Emmart). By means 
of fluorescein antibody, prolactin has been local- 
ized in the acidophilic cells of the anterior pitui- 
tary of the cat, rat and mouse (Drs. Emmart 
and Spicer in collaboration with Dr. Bates of 
NIAMD). 

Juxtaglomerular Apparatus 

In collaboration with Dr. G. Kaley of New York 
University, evidence has been accumulated to sug- 
gest that the renal granular juxtaglomerular cells 
are related to erythropoietin production in the 
rat. A direct relationship has been found between 
the secretory activity of these cells and the plasma 
erythropoietin levels. In rats the administration 
of cobalt, or exposure to hypoxia, or phenylhy- 
drazine, or oligenic shock results in a distinct ele- 
vation of both parameters. Also, rats that have 
been experimentally depleted of their juxtaglomer- 
ular cell granularity show a very poor response to 
erythropoietic stimuli (Dr. Demopoulos). 

Melanoma 

The tyrosinase inhibitor, DL-/?-phenyl lactic 
acid (PLA) , decreases oxygen consumption in cells 
of pigmented S-91 melanomas, but not in amela- 
notic cells of the same tumor which are devoid 
of tyrosinase. This inhibition of respiration, 
which can be demonstrated in a tyrosine- free medi- 
um is accompanied by a simultaneous increase in 
aerobic glycolysis. Since the specific PLA-sus- 
ceptible segment in melanotic S-91 cells is in- 
hibited by PLA in a manner which is unique and 
similar to the way PLA inhibits S-91 tryosinase, 
it is postulated that the former segment of cell 
respiration is attributable to tyrosinase. Growth 
studies, in vitro, correlate fairly well with the 
manomeric results. Phenyl lactic acid in levels of 
l-1.5mM/liter selectively prevent growth of mela- 
notic S-91 explants if present at zero time in their 
growth curve. Delayed addition of PLA to cul- 
tures that have incubated for more than two hours 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



159 



in normal medium is without effects. The selec- 
tivity of phenyl lactic acid is not as marked in 
tissue culture as it is manometrically since higher 
concentrations (100% increase over 1 mM) of this 
agent can affect growth of non-melanotic control 
tissues as well. The de-aminated analogues of 
phenylalanine can inhibit protein synthesis in 
vitro, in high concentrations, by blocking tyrosine 
incorporation. This may offer a clue to the non- 
specific growth inhibiting effects of high doses of 
PLA. This inhibition can also prevent growth 
of melanotic $-91 tumors freshly transplanted 
into young DBA/2 mice while growth of their 
somatic tissues is not impaired, but it has no effects 
on established tumors (Dr. Demopoulos). 

Metabolic Requirements for Cardiac Lesions in 
the Carcinoid Syndrome 

In an attempt to reproduce in experimental 
animals the cardiac lesions seen in some human 
patients with the carcinoid syndrome, long and 
short term metabolic studies with guinea pigs and 
rabbits have been undertaken. These animals 
have been subjected to a niacin and tryptophan 
deficient diet for varying time periods and exposed 
to liver damage and exogenous 5-hydroxy-trypt- 
amine, simulating the hepatic metastases and tryp- 
tophan deficiency of patients with the carcinoid 
syndrome having cardiac valvular lesions. A 
valvular endocarditis comparable in some respects 
to that seen in humans with the carcinoid syn- 
drome has now been produced. A further evalua- 
tion of the causative mechanism involved in this 
unique disease process is in progress (Dr. Spatz). 

Monoamine Oxidase Inhibitors 

Monoamine oxidase inhibitors have been widely 
used in the treatment of mental depression and 
hypertension. In a collaborative study with Dr. 
Maling of the NHI, it was found that prolonged 
administration in dogs of two of these inhibitors, 
phenylisopropylhydrazine (JB 516) or phenyl - 
isobutylhydrazine (JB 835), consistently pro- 
duced marked neurologic effects, including ataxia, 
tremors, nystagmus, and impaired motor coordi- 
nation. Neuropathology lesions involving the in- 
ferior olivary nucleus and less frequently the 
pyrif orm lobe, particularly the amygdaloid nuclei 
were seen in dogs but not in cats, rabbits or squir- 
rel monkeys. The lesions are not due directly to 



monoamine oxidase inhibition, since elevations in 
serotonin levels in the pyriform lobe and brain 
stem due to such inhibition were produced by 
other monoamine oxidase inhibitors in animals 
showing no such lesions (Dr. Highman). 

Pathology of Rheumatic Disease 

The role of the blood vessels in the histogenesis 
of the subcutaneous nodula of rheumatoid arthri- 
tis has been demonstrated graphically by a three- 
dimensional reconstruction of an early lesion. It 
was based on more than 500 serial sections of a 
nodule only 3 days old. The inflammatory proc- 
ess was found to center about damaged segments 
of the terminal vascular bed, including minute 
arteries, arterioles, capillaries and venules. The 
episodic and centrifugal character of the process 
also were apparent from the reconstruction. The 
observations are believed to lend support to the 
thesis that inflammation of minute blood vessels 
is an integral part of the pathogenesis of rheuma- 
toid arthritis. 

A new interpretation of the nature of the elas- 
ticity of articular cartilage and a method for in- 
vestigating its component elements have been 
developed in collaboration with Dr. S. M. Elmore, 
NHI, and members of the Instrument and Engi- 
neering Branch. The apparatus measures the 
deformation and recovery of articular cartilage 
during and following static loading under condi- 
tions of immersion. Older views of the plastic 
nature of cartilage were found to be based on 
tecchnical artefacts arising from drying of the 
tissues. The compressibility of the tissue was 
greatly affected by the ionic environment of the 
bath. The findings are of fundamental signifi- 
cance in the normal physiology of joints and are 
being pursued as a promising lead to understand- 
ing the pathogenesis of osteoarthritis (Dr. 
Sokoloff). 

In view of the continued use of gold compounds 
in t\\Q treatment of rheumatoid arthritis, it was 
thought desirable to apply the activation analysis- 
autoradiographic method as described in the obe- 
sity study to determine the distribution of gold in 
the joints of mice following gold administration. 
The findings suggested selective uptake of gold- 
thioglucose as well as goldthiomalate in areas of 
rapid bone renewal and in portions of the syn- 
ovium (Dr. Brecher). 



160 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Studies in Fine Structure 

Ultrastructural detail revealed by electron mi- 
croscopy and the localization of acid phosphatase 
activity are providing new insight into the mech- 
anisms of thyroxin mobilization by rat thyroid 
glands. Thyroid stimulating hormone (TSH) 
stimulation of hypophysectomized animals per- 
mits the observation of this phenomenon as a func- 
tion of time (Drs. Wetzel and Spicer in collabora- 
tion with Dr .Wollman, NCI) . 

Descriptive anatomical studies of the Turbel- 
larian genus Dugesia have been extended with 
particular emphasis on the excretory organs of 
this animal. An outstanding degree of regional 
cell specialization along the nephric unit clearly 
indicates a high degree of corresponding func- 
tional specialization in this phylogenetically prim- 
itive form (Dr. Wetzel). 

Collaborative studies with Dr. Mortimore on 
isolated, cyclically perfused rat livers indicate 
that over a period of one and one-half hours the 
fine structural integrity of hepatic parenchymal 
cells can be maintained while many physiological 
and biochemical parameters are accurately moni- 
tored and controlled. An absence of physiological 
concentrations of insulin from such a perfusate, 
for example, results in extensive disorganization 
of normally parallel arrays of rough surfaced en- 
doplasmic reticulum. A marked decrease in pro- 
tein synthesis and in amino acid incorporation by 
these experimental livers was observed concomi- 
tantly, thus providing additional support for the 
role of organized rough surfaced endoplasmic re- 
ticulum in the production of protein. The appar- 
ent lability of this system is of particular interest 
and will be further investigated (Dr. Wetzel). 

A study is under way on the nuclear and nucle- 
olar cytology of basic proteins with acid dyes and 
their modification by heavy metal salts with the 
objective of developing a differential cytochemical 
staining method for both light and electron mi- 
croscopy (Dr. Suskind). 

Factor 3 — Selenium 

The physiological significance of the element se- 
lenium was discovered several years ago (K. 
Schwarz and collaborators) when selenium was 
found to be the integral part of the so-called Fac- 
tor 3, which prevents dietary liver necrosis. Since 
then, 18 species have been found in which fatal or 



severe deficiency diseases can be prevented by trace 
amounts of selenium compounds. The so-called 
white muscle disease of selenium deficiency in 
sheep and in cows, is of economic importance. 

The rat bioassay has been used to determine the 
relative biopotencies of different selenium com- 
pounds. However, in recent studies by Dr. Schu- 
bert and collaborators (Oregon State University) 
y,y-diselenodivaleric acid, highly potent in the rat, 
was found to be relatively ineffective against white 
muscle disease in lambs. The result indicates that 
biopotencies of different selenium compounds may 
vary from species to species. 

Organoselenium compounds, synthesized by 
Prof. Dr. Arne Fredga at the Univ. of Uppsala, 
Sweden, in collaboration with this laboratory, has 
led to a number of new findings. Fatty acids con- 
taining selenium in place of a CH 2 group at var- 
ious distances from the carboxyl group show no 
difference in biopotency with the element at an 
even or uneven numbered position in the mole- 
cule (K. Schwarz and J. P. Stesney). The bio- 
potency of most of these compounds is similar to 
that of selenite selenium. However, the 9-selena- 
penta- and hexadecanoic acids, and the 12-selena- 
penta- and hexadecanoic acids, are substantially 
less effective than selenite, suggesting their pos- 
sible use as chemotherapeutic agents against 
protozoa. The most effective Factor 3-active 
selenium compound synthesized thus far is benzyl- 
seleno-<o-pelargonic acid, with an ED 50 of .8 ^g per 
100 g of diet. This is close to .7, the value 
originally found for natural Factor 3. 

Using Se 75 as a marker, five fractions containing 
the main selenium carrying components in urine 
have been isolated in amounts satisfactory for 
Factor 3 bioassay in the rat (E. Sweeney and R. 
Jacobs) . Application of more sensitive, new tech- 
niques of paper chromatography to the urine of 
rats after supplementation of radioactive selenite 
selenium has shown that there are about 15 differ- 
ent organoselenium components excreted. None 
of them seems to be inorganic. 

Since there are large differences in the selenium 
contents of various soil formations, the Factor 3 
potency of skim milk powders from various geo- 
graphical areas of the USA, put at our disposal 
by the Atomic Energy Commission, has been deter- 
mined by bioassay against dietary liver ne- 
crosis in the rat. While skim milk powder from 
some areas, notably Oklahoma and North Dakota, 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



161 



afforded almost complete protection when added 
to the diet at 5% levels, those from some other 
areas were without appreciable effect even at 15% 
levels. The Factor 3 activity was not influenced 
by the method used for making the dried milk. 

To establish the therapeutic index, the LD 50 for 
various selenium compounds was determined in 
rats raised and maintained under conditions iden- 
tical to those used in the Factor 3 bioassay pro- 
cedure (L. Hopkins). For selenite selenium, a 
ratio of ca 2000:1 was found between the LD 50 
when applied orally, and the 50% effective daily 
dose required to prevent necrotic liver degenera- 
tion. No direct correlation exists between Factor 
3 potency and toxicity. y,y-Diselenodivaleric acid, 
when compared to selenite selenium, was only 60 
percent as toxic as the latter, in spite of its higher 
biopotency in the Factor 3 assay. 

The selenium content of a variety of purified 
enzymes, many of them crystalline, varied from 
to 105 fig per g of enzyme. If stoichiometric levels 
of selenium were present in bound form, the ele- 
ment could be expected to amount to ca 1 mg, as- 
suming a molcular weight of the enzyme of ca 
80000. 

Respiratory Decline, Sulfhydryl Groups and the 
Mode of Action of Tocopherol 

In studies on respiratory decline (K. Schwarz 
and C. Lee), the metabolic impairment seen in 
prenecrotic livers of animals on necrosis produc- 
ing, deficient diets, it was ascertained that respira- 
tory breakdown and the protective effect of toco- 
pherol and other naturally occurring quinonoid 
substances is not related to the presence of sodium 
ions in the medium, or to the breakdown of ion 
transport mechanisms. The substrate specificity 
of respiratory decline and the protective effects 
of tocopherol were studied. The phenomenon oc- 
curs only with certain substrates of the Krebs 
cycle. Primarily involved are a-ketoglutarate, 
pyruvate plus malate, and also fumarate and 
malate. With these acids, 2 consumption de- 
clines by 70-90% in the absence of tocopherol. 
With succinate, as well as pyruvate alone, or 
citrate, losses of 2 consumption are less pro- 
nounced, and the protective supplements have no 
effect. 

Lipid peroxidation does not seem to be a pri- 
mary factor in respiratory decline, therefore, some 
more intricate mechanism of action, other than the 



antioxidant activity of tocopherol, is decisive for 
the understanding of the metabolic role of vitamin 
E in this system. 

Respiratory decline of vitamin E-deficient livers 
occurs equally well at any pH between 6.2 and 7.6 ; 
however, the potency of tocopherol in the system 
is strongly pH dependent. A sharp break in the 
response occurs below pH 7. Below 6.6, homog- 
enates do not respond at all. In the initiation of 
respiratory decline trace elements are involved, 
specifically by inactivating sensitive sulfhydryl 
sites (or similar sensitive loci). Complexing 
agents, such as EDTA, prevent the phenomenon. 
The pH dependency of the dose response to EDTA 
showed no significant difference from tocopherol. 

Various electron transport inhibitors were used 
to clarify whether the effect of protecting sub- 
stances on respiratory decline is correlated to spe- 
cific sites of the electron transfer chain. These 
studies led to the conclusion that the primary site 
of the metabolic impairment in respiratory de- 
cline is neither in the electron transfer chain, nor 
in oxidative phosphorylation, but in certain de- 
hydrogenase systems linking the Krebs cycle to 
the cytochromes. All substrates involved use 
DPN-dependent pathways. 

With a-ketoglutarate as substrate, the enzyme 
lipoyl dehydrogenase has been identified as a tar- 
get of the inactivation mechanism (K. Schwarz, 
C. Lee and J. A. Stesney). A close parallenism 
exists between the degree of respiratory failure 
and the loss of activity of this enzyme. Addition 
of tocopherol prevented the inactivation of the 
enzyme and at the same time prevented respira- 
tory decline. Lipoyl dehydrogenase is very sen- 
sitive to trace metals such as Cd ++ and Cu ++ ; it is 
inactivated by arsenite. The results indicate that 
the trace element sensitive site of lipoyl dehydro- 
genase is a point at which tocopherol exerts its 
protective effect. It is also apparent that under 
these dietary and experimental conditions, lipoyl 
dehydrogenase is rate limiting for 2 consump- 
tion when a-ketoglutarate serves as a substrate. 

The inhibitory effects of Cd ++ , and Cu"" and Cu + 
on a highly purified lipoyl dehydrogenase from 
heart muscle was investigated. With 2 or 4 
juatoms of Cu ++ per /miole of enzyme, ca 25% and 
70% inhibition were obtained, respectively. Ad- 
dition of a-tocopherol to the system afforded par- 
tial protection (ca 50%) against the Cu- inhibi- 
tion. Substances such as the Simon metabolite of 






162 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tocopherol, tocopherylquinone, medadione and 
also DPPD, produced similar results. These 
investigations open up new approaches to the 
mode of action of vitamin E and similar factors 
in intermediary metabolism, with particular bear- 
ing on the question of a functional interrelation- 
ship of quinonoid compounds to active sulf hydryl 
groups of enzymes. These groups, and possibly 
similar groups, such as selenol groups, appear to 
be the primary sites of attack, on the molecular 
level, by deleterious agents producing degenera- 
tion and necrosis. 

Studies on lipoic acid levels in livers of animals 
on various diets (in cooperation with V. Dewey, 
Amherst College) showed: In animals on the basal 
Torula diet, deficient in sulfur amino acids, lipoic 
acid levels amounted to only approximately 60% 
of those from control animals on normal diets. 
The supplementation of (selenium-free) sulfur 
amino acids raised the depressed lipoic acid levels 
to normal. Other agents preventing liver necrosis, 
namely vitamin E and Factor 3-selenium, were in- 
effective when used separately. However, the 
combination of selenium and tocopherol brought 
the lipoic acid level almost back to that found in 
controls. Hence, the delaying effect of sulfur 
amino acids on liver necrosis may be mediated by 
an effect on the tissue levels of lipoic acid, a sulfur 
containing cofactor synthesized within the or- 
ganism. 

Glucose Tolerance, Chromium (III) and Other 
Factors 

In view of the role of trivalent chromium as a 
dietary factor (glucose tolerance factor) neces- 
sary for normal utilization of glucose (K. Schwarz 
and W. Mertz), the uptake of trivalent, radioac- 
tive chromium 51 has been investigated in rats (L. 
Hopkins and C. Cisar). With various levels of 
supplementation, a ceiling for chromium uptake 
through the gastrointestinal wall seems to exist, 
indicating a specific mechanism for chromium 
(III) absorption. Similarly, a specific mech- 
anism for chromium (III) transport in blood is 
found in the /^-globulin fraction. 

Glucose tolerance is known to be affected by a 
variety of dietary factors other than chromium 
(III). In animals maintained for extended 
periods on Torula yeast diets, which are deficient 
in methionine and tryptophan, impaired rates of 
glucose uptake were found which did not respond 



to chromium. Normal rates of glucose removal 
were re-established by dietary supplementation 
with tryptophan. 

OFFICE OF MATHEMATICAL RESEARCH 

Work has continued within the areas and along 
the lines indicated in the summary of last year. 
These are: Mathematical and computational 
methodology for mathematical models and de- 
tailed study of iodine metabolism in the thyroid 
system; mathematical formulation and analysis 
of the model for a dendritic neuron ; general math- 
ematical problems arising from the rate behavior 
of metabolic systems; mathematical studies of 
visual perception. In addition, two new areas 
have been included. These are : Theoretical stud- 
ies of the kinetic, thermodynamic and statistical- 
mechanical properties of macromolecules ; and 
studies on the numerical solution of integral equa- 
tions, with special reference to convergence of iter- 
ative techniques and bounds for error, employing 
the methods of functional analysis. 

The summaries below are in terms of "high 
lights" or examples. 

The computer program developed for the an- 
alysis of kinetic data has been expanded to include 
reaction kinetics of zeroeth, first and second orders. 
The computational methods have been improved 
to accelerate convergence. Applications of the 
program to a variety of biomedical problems have 
increased at NTH. In addition, the program has 
also been put into operation at the data processing 
research centers of the Veterans Administration 
in Washington, D.C., and Los Angeles, Calif., The 
Brookhaven National Laboratories, Upton, N.Y., 
and the Lawrence Radiation Laboratories in 
Berkeley, Calif. Close collaboration with these 
centers is maintained. Several groups outside 
NIH have also established liaison with the group 
for the use of the program. A study in collabora- 
tion with Drs. R. Bellman of the Rand Corpora- 
tion and Mr. Elkind of the NCI for the develop- 
ment of a mathematical-physical model for the 
interpretation of data on the dynamics of irradi- 
ated cell populations has been undertaken. The 
efforts of this past year went into the establish- 
ment of communications between the various 
points of view and the formulation of the prob- 
lem. The theoretical testing of the first postulated 
model has started. It is hoped that the theoretical 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



163 



model evolved will be sufficiently general to be 
useful in other areas of mathematical biology as 
well. Data analysis for the kinetics of iodine in 
patients of various abnormalities has continued. 
A working model has now been derived in terms 
of which most of the data has been satisfactorily 
analyzed. An evaluation of the results of this 
analysis is presently under way ( Dr. Mones Ber- 
man and Mrs. Marjory Weiss). 

The mathematical model of a dendritic neuron 
has been developed further, and computations are 
in progress to explore its implications for several 
problems of neurophysiological interest. By rep- 
resenting a dendritic tree as an approximately 
equivalent compartmental system, it has become 
possible to explore the theoretical consequences of 
various spatio-temporal patterns of synaptic ac- 
tivity on a dendritic tree. Computational explo- 
ration of this problem has been greatly facilitated 
by the use of a computer program developed by 
Berman, Shahn and Weiss. Considerable time 
and effort have been given to developing and test- 
ing computer programs for use on the Honeywell 
800. These programs include (a) computations 
of somadendritic spread of core current and mem- 
brane potential in dendritic trees of finite extent, 
(b) computations of extracellular potential dis- 
tributions for various sequences of dendritic ar- 
rangements. By means of these computer pro- 
grams it is possible to compute extracellular 
action potentials for pyramidal cells which are ar- 
ranged in cortical layers. Such calculations are 
being undertaken in collaboration with Dr. Gordon 
Shepherd (NINDB, Associate Member OMK), 
the results of such calculations will be compared 
with experimental results which he has obtained 
from the olfactory bulb of cats. A procedure has 
been developed (and programmed for the Honey- 
well 800) for the estimation of surface areas and 
dendritic input conductance of neurons from in- 
complete histological information. It is possible 
to demonstrate that even histological sections of 
200 micron thickness, as prepared by outstanding 
neurohistologists, frequently display as little as 
25 percent of the estimated total dendritic surface. 
This procedure is now being applied to a set of 
histological measurements made by Dr. Aitken of 
University College, London (Dr. Wilfrid Ball). 



The theorem on a "mammillary matrix" of the 

form -p. where r and c are a row and column 

of n elements and D is diag(di, d 2 , . . ., d n ) has 
been extended. It has been shown that if X is a 
repeated root of the matrix, then \=di for some i; 
that some dj is a root if and only if for some suita- 
ble indexing d! = d 2 = . . . = d r = d and then d is a 

root of multiplicity r=l. The exponential com- 
ponent exp(dt), corresponding to a root of D 
which is a root of the matrix, is always absent from 
the central compartment and is absent from the 
peripheral compartments if certain initial condi- 
tions in them (including the commonly employed 
zero condition) are satisfied. This lumping effect 
depends upon the identity of some sub-set of the 
di and not, as in the classical case, upon identical 
compartments. The properties of the mammillary 
matrix have proven to be of use in areas having 
nothing to do with compartmental analysis. For 
example, they can be applied to stability studies of 
the linearized Hodgkin -Huxley equations. In 
particular it has been shown that if the quantity 
H( A) =a — rD _1 c is positive, the system is unstable ; 
if H(A)<0 and -H(A)>Q=-2(r,-c 1 ) 2 /d,>0 
the system is stable; if Q> — H(A)>0, the system 
may be stable or unstable. Thus only the cases 
in which H(A) lies between zero and — Q need be 
investigated in numerical detail. The relation 
between the column sums of a matrix and those of 
its adjoint and the applications reported last year 
have been shown to be a special case of tht 
theorem: If v is an eigenvector of A, vA = Xv, 
then v adj(A— xl)=/xv, where ju= |A — XI |(X— x), 
is true for arbitrary x and relates the roots of A 
and adj(A — xl) even in the singular case. An 
extensive series of theorems on linear systems, 
most of which are new, with interpretation and 
applications have been collected in an article to 
appear in the Ann. N.Y. Acad. Sci. (Dr. John Z. 
Hearon). 

The basic model for brightness perception, 
reported last year, has been refined. Studies of 
published experimental data on Mach bands led 
to the conclusion that the visual system is 
markedly non-linear in its spatial characteristics. 
This conclusion is contrary to that drawn by the 
authors who published the data and is based upon 
a simple but more sensitive test for non-linearities. 



705-685 — 63- 



-12 






164 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Since Mach bands are a local effect and must, in 
any model, be produced by a local averager, it can 
now be asserted that local averaging is non-linear, 
consistent with the overall non-linear character of 
the original basic model. Extension of the basic 
model to the time domain has begun with a study 
of nicker response and fixed retinal image effects 
(Mrs. R. B. Marimont, NIMH, Associate Mem- 
ber, OMR). 

Consideration has been given to the estimation 
of the equilibrium extent of polymerization of 
double stranded nucleic acids as they arise from 
nucleoside triphosphates as monomer sources. It 
has been found that the free energy of formation of 
a phosphodiester bond by a reaction represented 

by XTP+X <=± XPX + PP is about 

zero. However, when there is a moderate thermo- 
dynamic driving force for strand association high 
degrees of polymerization may be anticipated at 
equilibrium. A matrix method for treating the 
copolymerization statistics of certain types of 
systems has been extended to allow for the influ- 
ence of remote interactions in the chain. By use 
of the matrix calculus, certain average properties, 
(e.g. degrees of polymerization, numbers of specific 
sequences of units, etc.) of the copolymeric chain 
can be computed. The possible application of 
this approach to nucleotide copolymers is under 
investigation (Dr. Leonard Peller). 

Work on a method to solve numerically the 
linear Fredholm integral equation, (1): f(x) — 



J' 



K(x,y)f(y)dy = g(x), was initiated in collabora- 
tion with Dr. P. A. Anselone at the Mathematics 
Research Center, U.S. Army, University of Wis- 
consin. Here K(x,y) may have, on the rectangle, 
a^x, y^b, weak discontinuities in the sense of 
Kolmogorov and Fomin. We convert (1) into (2): 

f(x)- C f b K(x,t)K(t,y)f(y)dtdy=g(x) 

Ja J a, 

+ K(x,y)g(y)dy 

Ja 

and approximate the kernel 

K (2) (x,y)=f b K(x,t)K(t,y)dt 
by the Gauss quadrature (3): 

K„ <2) (x,y)-f:K(x,t k < n >)K(t ![ (n >,y)w k (n) 

k = l 



thus replacing the kernel of (2) by an approximat- 
ing kernel (3) of finite rank. This work has been 
continued here. Now it can be proved that the 
sequence of approximating operators (I — K n (2) ) _1 
converges, in the norm sense, in the Banach space 
of bounded linear operators to the operator 
(I — K (2) ) -1 and that the sequence of approximat- 
ing solutions converges in the norm to the solution 
in the space of functions continuous on the interval 
[a,b]. A general formula for the error estimate has 
been developed and its practical aspects explored 
on the computer. This work is sufficiently general 
to include, with numerical feasibility, most appli- 
cations and in particular those involving the 
Volterra type integral equation to indicator dilu- 
tion techniques in hemodynamics (Dr. Jose M. 
Gonzalez-Fernandez ) . 

CLINICAL INVESTIGATIONS 

A total of 529 inpatients was admitted during 
the 12-month period from December 1, 1961 to 
November 30, 1962, an increase of 49 patients 
(10%) over the same period last year. The total 
patient days was 19,423, a decrease of 433 over the 
preceding year. The average inpatient stay at the 
Clinical Center was 37 days. The average census 
was 53 (76%). In the Admissions and Followup 
Department 2,041 patients were examined and 
studied, an increase of 160 over the past year. 

Investigations conducted in the laboratories of 
clinical investigation of NIAMD have resulted in 
109 publications in scientific journals, mono- 
graphs, annual reviews and medical textbooks. 
Dr. Harold Edelhoch was awarded the Van Meter 
Prize by the American Goiter Association in rec- 
ognition of his original work on the development 
of a spectrophotometric method (not requiring 
hydrolysis of the protein) for the analysis of tyro- 
sine, monoiodotyrosine, diiodotyrosine and thy- 
roxine in native or iodinated thyroglobulin. Dr. 
Paul A. di Sant'Agnese received an honorary 
degree of Doctor of Medicine at Justus Liebig 
University, Giessen/Frankfurt, West Germany 
for his contributions to our knowledge of cystic 
fibrosis of the pancreas. 

ARTHRITIS AND RHEUMATISM BRANCH 

Association of Sjogren's Syndrome With Malig- 
nant Lymphomas 

Clinical investigation on a series of 60 patients 
with Sjogren's syndrome admited to NIAMD over 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



165 



the past 4 years led to the observation that these 
patients have an unusual abundance of abnormal 
circulating antibodies to tissue components. For 
example, every patient had rheumatoid factor 
(whether or not they had rheumatoid arthritis). 
The majority had antinuclear factors, about one- 
half had complement-fixing antibodies to diverse 
organs and tissues that were not species specific 
and about one-fourth had thyroid antibodies. 
During the past year four of our patients de- 
veloped lymphomas ; three had reticulum cell sar- 
comas and one a lymphoma resembling Walden- 
strom's maeroglobulinemia. A fifth case of 
Sjogren's syndrome with reticulum cell sarcoma 
was found in the literature. We learned of a 
sixth case at the Mayo Clinic complicated with 
Hodgkin's disease (personal communication). 

The unusual association in the same patient 
of these two uncommon diseases cannot be ascribed 
to mere coincidence. The first five patients share 
certain clinical and laboratory features which dis- 
tinguish them from the usual patient with 
Sjogren's syndrome. Chief among these are 
splenomegaly, purpura, vasculitis, leukopenia, 
lymphopenia, the presence of rheumatoid factor, 
antinuclear factor, antithyroglobulin, comple- 
ment-fixing antibodies and immune globulins. 
With the aid of serologic and immunochemical 
methods, particularly immunoelectrophoresis, 
these abnormalities in the 7S gamma globulins, 
/? 2 A and /? 2 M globulins are being examined more 
closely. Hypogammaglobulinemia is present in 
two patients and diffuse hypergammaglobulin- 
emia in two. These alterations in immune 
globulins correlate well with other features of the 
disease and may reflect a basic abnormality in 
immunologic responsiveness. 

It may be important to note that three of our 
four patients had received deep X-ray therapy to 
the enlarged parotid glands (part of Sjogren's 
syndrome) prior to developing lymphomas (Drs. 
Bunim and Talal) . 

Factors That Direct Organ Distribution of 
Lymphocytes 

Preliminary studies have confirmed the fact that 
living, isotopically labelled (P 32 ) lymphocytes are 
concentrated in the spleen within 30 minutes after 
intravenous injection and that heat-killed lym- 



phocytes are not. Preincubating lymphocytes 
with a partially purified preparation of glycosi- 
dases obtained from Clostridium welchii pre- 
vented the accumulation of lymphocytes in the 
spleen, though this treatment did not kill the cells 
(as determined by vital staining) . The inhibition 
of accumulation of homologous lymphocytes in 
spleen by glycosidases was exponentially propor- 
tional to enzyme concentration and directly pro- 
portional to time of incubation. This enzyme 
effect could be prevented by the prior addition of 
simple sugars (galactose, mannose, fucose, N- 
acetyl galactose-amine, N-acetyl glucose -amine) to 
the reaction mixture. However, the enzyme effect 
could not be inhibited by glucose added in the 
same amounts as the other sugars. The sugars 
which were found to inhibit the enzyme effect 
were shown to be present in lymphocyte membrane 
preparations. One possible explanation for the 
concentration of lymphocytes in the spleen is that 
there are complementary binding sites in the 
spleen for the macromolecular surface structure 
of intact lymphocytes and that this specific surface 
structure of the lymphocyte is determined at least 
in part by surface polysaccharide (Drs. Gesner and 
Ginsburg) . 

Antigenic Composition of Glutamic 
Dehydrogenase 

Mammalian glutamic dehydrogenase (GDH) is 
a high molecular weight tetramer that is able to 
dissoicate reversibly under the influence of diethyl- 
stilbestrol (DES) into monomeric units of differ- 
ent amino acid specificity. It can be further and 
irreversibly dissociated by the detergent sodium 
dodecylsulfate (SDS) into 16 subunits of molec- 
ular weight approximately 60-70,000. Rabbit 
antisera have been prepared against these two dif- 
ferent forms of mammalian GDH. Electrophore- 
tic, immunoelectrophoretic and agar diffusion 
studies have been performed in 1% agar employ- 
ing a normal buffer at pH 8.2. Glutamic dehy- 
drogenase activity can be identified even after 
immunologic precipitation in agar by means of 
specific biochemical reactions leading to a colored 
product. 

The dissociation of GDH by DES or SDS can 
occur during electrophoresis or diffusion in agar. 
Therefore, electrophoretic and immunochemical 



166 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



methods can be employed to study the protein, bio- 
chemical and antigenic nature of the molecular 
forms involved in these dissociations. 

Preliminary investigations indicate that the 
three molecular forms studied (tetramer, mono- 
mer and SDS fragment) have different and char- 
acteristic electrophoretic mobilities and antigenic 
properties (Drs. Talal, Yielding, and Tomkins 
and Mr. Mushinski). 

Effect of Steroid Hormone on Structure and 
Function of Enzymes {in Vitro) 

During 1962 our conclusions concerning the rela- 
tionship between physical structure and catalytic 
activity of GDH have been confirmed by inde- 
pendent means of measurements. It has now be- 
come evident that control of this enzyme is a rather 
specific process and apparently involves specific 
sites on the enzyme molecule which are concerned 
with control. Furthermore, it appears that rather 
specific SH groups are involved with these control 
sites in contrast with the sites concerned with catal- 
ysis of the chemical transformations. These con- 
clusions, although derived from our previous in- 
vestigation, have now been made more definite by 
the following confirmatory lines of evidence. 

1. Correlation of steroid induced changes in the 
structure of GDH with changes in its function : 
Studies on the correlation of the physical state of 
the GDH molecule and its catalytic activity have 
been continued in collaboration with Dr. Gordon 
M. Tomkins. It has now been possible to study 
the effects of estrogenic hormones and various ex- 
perimental conditions on both the physical state 
of the GDH molecule and its ability to catalyze 
the glutamic dehydrogenase and the alanine de- 
hydrogenase reactions using precisely the same re- 
action mixture for the kinetic studies and the 
physical studies. The molecular weight in these 
experiments has been determined by light-scatter- 
ing, and it has been possible to show a direct cor- 
relation between those two parameters — confirm- 
ing the previous conclusion that the most 
aggregated form of the molecule catalyzes the glu- 
tamic dehydrogenase reaction and the disaggre- 
gated form catalyzes the alanine reaction. Fur- 
thermore, ability of the steroid hormones to 
dissociate the enzyme molecule in to subunits is 
exactly correlated with inhibition of glutamate 






and activation of alanine activities. Light-scat- 
tering experiments have also confirmed that the 
molecule is reaggregated by ADP, L-leucine and 
various amino acids, and that the substrates of 
the GDH reaction have profound effects on the 
physical state of the enzyme molecule. While 
making an attempt to determine the dissociation 
constant for the enzyme under various experimen- 
tal conditions, it has become apparent that the dis- 
sociation of the tetrameric form into monomeric 
subunits involves one or more intermediate forms 
of the enzyme such as dimer or trimer. It is ap- 
parent that the various experimental circumstances 
which change the apparent weight average molec- 
ular weight of the molecule simply shift the disso- 
ciation constant of the overall reaction toward 
either the more dissociated form or the more ag- 
gregated form. 

It has also been possible to show both in this 
laboratory and in Dr. Tomkins' laboratory that 
the specific activity as well as the molecular weight 
of the purified enzyme toward alanine and gluta- 
mate varies with enzyme concentration in a manner 
predicted for a dissociating system in which the 
aggregated form of the enzyme catalyzes the glu- 
tamic — and the dissociated form the alanine de- 
hydrogenase reactions. 

The interrelationship of the alanine and glu- 
tamic dehydrogenase activities of the GDH mole- 
cule have also been examined in tissue culture cells 
and it has been observed that sonicates of Hela 
cells have predominately alanine activity and must 
be activated with ADP to exhibit significant GDH 
activity. 

2. Role of -SH groups in the control and activity 
of GDH : Experiments on the different sulfhydryl 
groups in the enzyme have also been continued, 
particularly in relation to control of enzymic func- 
tion. The previously described ability of para- 
chloromercuribenzoate to prevent the steroid in- 
hibition and the ADP activation of the enzyme has 
been amplified by the additional finding that meth- 
ylmercuric bromide and methylmercuric chloride 
are even more specific in preventing the control of 
the enzyme by steroid, leucine, ADP, detergents 
and DPNH while not producing any inhibition of 
the reactions themselves. N-ethyl maleamide, on 
the other hand, has no effect either on the catalytic 
properties or on the sensitivity of the enzyme to 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



167 



control by other substances, but stabilizes the 
enzyme. It would appear that the SH groups in or 
near the control sites of the enzyme are somewhere 
intermediate between the most accessible SH 
groups and those which are remote within the 
structure of the enzyme molecule. In view of a 
recent report to the effect that parachloromercuri- 
benzoate has a capacity to disrupt GDH into sub- 
units without inhibiting its activity, it became of 
further interest to make a study of the effects of 
this compound on the molecular size of the enzyme. 
It was observed that when the enzyme was mixed 
with PCMB (or PCMPS) alone, the SH reagent 
did, indeed, disrupt the enzyme into subunits. 
However, when an identical experiment was run in 
the presence of substrates for the reaction, the dis- 
ruptive effect of the mercurial was prevented ex- 
cept at very high concentrations, when inhibition 
could also be observed. Using the ultracentrifuge 
and light-scattering, the "shielding" effect of the 
mercurial was shown to be mediated by its ability 
to prevent changes in molecular size induced by the 
various reagents. 

Studies on the nature and function of -SH 
groups in the enzyme have been continued. It 
has been shown that in addition to this ability of 
various mercurials to antagonize the effects of 
various "control" substances by binding to specific 
sites on the enzyme, they also interfere with bind- 
ing of DPNH as shown by fluorescence enhance- 
ment. This permitted a reexamination of our pre- 
vious conclusion that the DPNH binding shown by 
fluorescence enhancement did not represent the 
catalytically active cofactor, but other binding 
sites on the enzyme molecule. Accordingly, methyl 
mercuric bromide (or chloride) was shown to de- 
crease the affinity for DPNH as shown by fluores- 
cence enhancement while not affecting the Km for 
DPNH in the glutamic dehydrogenase reaction. 
It has also been shown that the shielding effect 
described previously can be obtained with a molar 
ratio of mercurial to enzyme as low as 10:1, thus 
indicating rather great specificity of the -SH 
groups involved. Recent data indicate that the 
ability of mercurials to stimulate GDH at pH 9 
(originally described by Hellerman) are also re- 
lated to an effect on the state of aggregation of the 
molecule (Drs. Yielding, Tomkins and Bitensky). 



Demography of Rheumatic Diseases: Genetic 
and Environmental Influences 

Several population surveys, described in 1961 
report, on the prevalence of rheumatoid arthritis 
(R.A.), osteoarthrosis (O.A.) and rheumatoid 
factor (RF) in the random population of the 
United States and in the Blackfeet Indians, have 
been continued in 1962. In addition, at the sug- 
gestion and invitation of Dr. Leonard T. Kurland 
of the Epidemiology Section of NINDB who has 
been conducting surveys on several of the Marianas 
Islands for amyotrophic lateral sclerosis and Park- 
insonism, about 500 serum samples from natives 
living on four of the islands were analyzed for uric 
acid concentration and for RF. As anticipated, 
the results were of unusual interest. 

1. Population Survey of the Blackfeet Indian 
Tribe 

The first phase of a two-part survey on the oc- 
currence of arthritis and RF in North American 
Indians was completed in northern Montana 
(Blackfeet tribe). The second phase will be con- 
ducted in southern Arizona Pima tribe) in 1963. 
A total of 1,103 or 85.8% of the registered Black- 
feet Indians residing on the reservation in north- 
ern Montana were examined clinically, serologi- 
cally and radiologically in 2 mobile clinics. In 
addition, the ABO secretor status was determined 
in 822 and blood types in 99. Analysis has not yet 
been completed but preliminary results on the first 
1,059 Indians examined reveal a prevalence of 
rheumatoid factor (bentonite flocculatlon test) in 
5.7% and of "probable" and "definite" rheumatoid 
arthritis in 4.0%. 

Blackfeet Family Study : In order to determine 
whether the occurrence of R.A. or RF was influ- 
enced by genetic factors two types of analyses 
were employed. The first consisted of determin- 
ing the occurrence of R.A. and RF among the first- 
degree relatives (parents, siblings and children) 
above age 29 of four classes of probands selected 
from the Blackfeet tribe: (1) seropositive rheu- 
matoid arthritis, (2) seronegative rheumatoid ar- 
thritis, (3) individuals who gave a positive test 
for rheumatoid factor but who had no arthritis 



168 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



and (4) probands negative for both rheumatoid 
arthritis and rheumatoid factor. No family ag- 
gregation of either rheumatoid factor or arthritis 
was demonstrable among the 390 first-degree rela- 
tives of 131 probands examined. The failure to 
demonstrate family aggregation is in contrast to 
the results reported by a number of contemporary 
workers in this field, both abroad and in the 
United States. The discrepancy is due largely to 
the nature of the controls chosen in the various 
studies. The second type of analysis used to deter- 
mine genetic linkage was by applying Penrose's 
formula for sibling pairs. All possible pairs of 
siblings in the entire population were studied and 
the number of pairs with both, one or neither 
member affected was determined and compared 
with the number that could be expected by chance. 
Again there was no evidence of significant aggre- 
gation in the kindreds. 

Finally, a similar analysis of the occurrence of 
RF and R.A. in spouses of subjects with RF and/ 
or R.A. was done in order to evaluate the possi- 
bility of a household environmental factor which, 
if operative, would result in an increased fre- 
quency of RF or R.A. However, no such influ- 
ence was detected. 

Our study has, therefore, failed to confirm the 
presence of a familial aggregation of rheumatoid 
arthritis or rheumatoid factor such as could be 
caused by a genetic factor or a household environ- 
mental factor as reported by other investigators. 

2. Marianas Islands Survey 1 

The field unit of NINDB has collected and sent 
frozen serum samples to us from 561 natives, age 
40 and over. BFT's for rheumatoid factor have 
been done on all and uric acid levels have been 
determined on 447 individuals. The frequency of 
positive BFT and hyperuricemia differed mark- 
edly on the different islands and races of people. 
The prevalence of hyperuricemia in this popula- 
tion was 30.1% of 186 men tested and 31.9% of 
207 women tested. A careful, well-designed sur- 
vey may determine the basis for this phenomne- 
non — genetic or environmental. Uric acid con- 
centrations determined on serum samples of 817 
enlisted men in the U.S. Army by Dr. Stetten's 
laboratory several years ago revealed that 2.5% 
had hyperuricemia (above 6.9 mg% by uricase 
spectrophotometric method). 



The prevalence of RF in Marianas natives was 
3.5% among 254 men tested and 3.9% among 307 
women tested. It will be noted that the Chamor- 
ros on Rota (both males and females) and the 
Carolinian women on Saipan have an unusually 
high frequency of positive BFT's. The values for 
these three groups are 13.2%, 8.7% and 10.7%. 

As will be seen (vide infra) the occurrence of 
positive BFT's in random population of the 
United States (above age 17) is 3.6%. There are 
several possible explanations for the high preva- 
lence in certain groups of Marianas natives. A 
careful survey may reveal the basis for this ob- 
servation. 

3. National Health Examination Survey 

The field survey phase of the National Health 
Examination described in last year's report was 
completed on December 1, 1962. Nearly 7,000 
adults (18 through 79 years) from 42 areas in the 
United States have had clinical examinations, 
radiographs of the hands and feet and serum col- 
lected for analysis for rheumatoid factor. Bento- 
nite flocculation tests are done in our laboratory. 
Thus far (December 1, 1962) we have analyzed 
6,669 serum samples collected from 41 areas. Of 
these, 237 or 3.6% gave a positive test. Examina- 
tion and interpretation of X-ray films of hands 
and feet have been completed on 2,397 persons 
from 15 areas. Of these, 19 or 0.8% had X-ray 
changes compatible with rheumatoid arthritis. 

Correlation of clinical findings have only been 
completed in 494 individuals from 3 areas. Six 
or 1.2% of these had a positive BFT and 12 or 
2.4% met the A.R.A. criteria for probable or defi- 
nite rheumatoid arthritis. Forty-four or 8.8% 
had moderate to severe changes of osteoarthritis 
on X-ray films of the hands and feet. All of these 
were 35 years of age or older (Drs. Burch, O'Brien 
and Bunim). 

Clinical Trial of Antirheumatic Drugs in Rheu- 
matoid Arthritis, Psoriatic Arthritis and 
Lupus Nephritis 

1. Hydroxychloroquine in Rheumatoid Arthritis 

Hydroxychloroquine has been evaluated by the 
Committee on Cooperating Clinics of the Ameri- 
can Rheumatism Association in a 6-month double- 
blind study. Arthritis and Rheumatism Branch 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



169 



of NIAMD participated in this project with the 
Rheumatology Service of the Georgetown Medical 
Division of the D.C. General Hospital, contribut- 
ing 14 patients to the study. In all, 121 patients 
were admitted to this double-blind trial, of which 
110 finished the study. There were 41 males and 
80 females in the group with the median age of 
53 years ranging from 31 to 72 years. All patients 
have had rheumatoid arthritis for more than 1 
year. Results of the study were reported in the 
Bulletin on Rheumatic Diseases, October 1962. 
The double-blind trial indicated moderate effec- 
tiveness of hydroxychloroquine in the treatment of 
rheumatoid arthritis. The five parameters evalu- 
ated included morning stiffness, grip strength, 
sedimentation rate, total number of joints involved 
with the arthritic process and time required to 
walk a distance of 50 feet. Morning stiffness 
showed improvement in 66% of the drug-treated 
patients and 54% of the placebo-treated patients. 
Number of joints involved decreased in 70% of 
the treated patients and in 62% of the control 
group. Grip strength improved in 86% of the 
drug patients and in 70% of the placebo patients. 
Improvement in walking time was similar in the 
two groups. The sedimentation rate showed im- 
provement in 64% of the drug patients and in 
45% of those receiving placebo. In no single pa- 
rameter mentioned was there a significant differ- 
ence between the placebo and drug groups. When, 
however, scores were assigned to each parameter 
and the patients rated on a range of to 5 accord- 
ing to the number of parameters that indicated 
improvement, there was a statistically significant 
difference between the patients treated with hy- 
droxychloroquine and those treated with placebo. 
Seventy-five percent of the drug-treated patients 
had significant improvement, whereas only 54% 
of the placebo group showed such favorable 
change. No serious side effects were observed dur- 
ing the trial period. 

2. High Dosage Corticosteroid Therapy in Lupus 
Nephritis 

Reported studies by Robert Kark and others 
have indicated that the administration of high- 
dosage corticosteroids (equivalent to prednisone, 
50 mg. daily) may be effective in prolonging the 



life of patients with nephritis due to lupus ery- 
thematosus. A study has been instituted in the 
A&R Branch of NIAMD to evaluate this form of 
therapy for this clinical problem. Patients ad- 
mitted to this study must have definite lupus ery- 
thematosus with a positive LE preparation and at 
least two major manifestations of SLE. These 
patients are subjected to percutaneous renal bi- 
opsy. When the biopsy specimen indicates the 
presence of lupus nephritis, prednisone is adminis- 
tered in a dosage of 50 mg. daily. The patients 
are subjected to a second renal biopsy after three 
months. If the clinical state or the biopsy speci- 
men indicates improvement, the steroid dose is 
then tapered to a lower maintenance level and the 
patient is observed at three-month intervals. Re- 
peat biopsies are performed at intervals of 3-6 
months in order to further assess the course of the 
disease. To date, six patients have had at least 
two renal biopsies. All patients had arthritis and 
clinical evidence of renal disease. In addition to 
these six, one patient was observed with clinical 
manifestations of nephritis without the biopsy 
procedure. Three of the seven had elevation of 
BUN prior to the steroid regimen. In each of 
these three the BUN levels became normal during 
the administration of steroids. One patient died 
in pulmonaiy edema after 1 month of therapy ; the 
BUN immediately prior to death increased above 
100 mg%. PSP excretion was also measured in 
these seven patients. In one case the value was 
abnormal prior to the steroid treatment and re- 
mained abnormal throughout the study period. 
In one other case a previously normal value fell 
to abnormal levels at the end of the study period 
even though the patient remained clinically well 
and the biopsy showed improvement. Side effects 
of the high dosage steroid regimen included peptic 
ulceration in one case, compression fracture of a 
vertebra in one, mild diabetes controlled with diet 
alone in two instances, psychic abnormalities in 
four, thrombophlebitis in one, mild hypertension 
in five, and edema formation in four. One patient 
developed posterior subcapsular cataracts, having 
had high doses of steroid prior to entrance in the 
study. It is planned to continue this study (Drs. 
Black, Bunim, O'Brien, Buchanan, Talal, Cohen 
and Alepa). 






170 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The Enzymatic Defect in Histidinemia, Metabo- 
lism of Aromatic Amino Acid and Homo- 
gentisic Acid in Phenylketonuria, and 
Ochronosis 

1. Phenylketonuria 

The micro method developed to measure the 
concentration of phenylalanine in blood has been 
used to determine how constantly the level is main- 
tained during the day by a phenylketonuric pa- 
tient on a diet very low in phenylalanine and how 
the level changes after a single oral dose (1 gm.) 
of L-phenylalanine. Finger-prick blood samples 
taken every 2 hours showed that very little varia- 
tion occurred during the day on the special diet, 
but the level increased within 2 hours after the 
ingestion of phenylalanine and remained elevated 
for at least 8 hours. More data of this type is 
needed to evaluate the factors (and their dura- 
tion) which alter the phenylalanine level in the 
blood of phenylketonurics, such as illness, fever, 
dietary changes, etc. Since patients on the special 
diet are tested rather infrequently, the interpre- 
tation of the values for dietary control purposes 
requires that these factors be recognized and 
evaluated. 

The method to measure phenylalanine has been 
modified so that blood and urine histidine concen- 
trations can also be determined and the modified 
method was used in our studies on the family with 
histidinemia. 

We have continued to analyze blood samples for 
phenylalanine for groups outside NIH as a con- 
firmatory test in cases suspected of being phenyl- 
ketonurics because of a positive ferric chloride 
test in the urine. The latter test is not specific for 
phenylpyruvic acid. Each time we have found a 
normal phenylalanine blood level, we have sug- 
gested that the urine be retested, and if it remains 
positive, that we be sent both urine and blood 
samples. This procedure led us to the finding of 
two cases of histidinemia since in this new meta- 
bolic disease one of the histidine metabolites ex- 
creted in the urine also gives a positive ferric 
chloride test. 

2. Histidinemia 

Clinical and biochemical studies have been car- 
ried out on a family in which two sibs have his- 
tidinemia. It has been demonstrated that the 
enzymatic defect in this metabolic disease is the 



absence of histidase. As a consequence, blood 
levels of histidine are greatly elevated and histi- 
dine and imidazolepyruvic acid are excreted in 
the urine. The affected sibs were also found to 
lack urocanic acid in the skin and sweat. They 
both also have speech defects but normal intel- 
ligence ; however, it is not certain that the speech 
defect is related to the metabolic disease. 

3. Experimental Ochronosis and Ochronotic Ar- 
thritis 

Our investigations have continued on the chem- 
ical and enzymatic steps involved in the forma- 
tion of ochronotic pigmentation of connective 
tissues characteristic of alcaptonuria, and its 
relationship to ochronotic arthritis. Earlier stud- 
ies dealt with the tissue distribution and binding 
of homogentisic acid and benzoquinoneacetic acid 
(the oxidation product of homogentisic acid). 
Homogentisic acid was found to be bound by 
physical forces to connective tissues, but benzo- 
quinoneacetic acid reacts chemically with these 
tissues. Homogenates of skin and cartilage in- 
cubated with the latter acid yield brown products 
resembling the ochronotic pigmentation of al- 
captonuric tissues. 

The binding of benzoquinone by connective tis- 
sues has been studied in more detail. No signifi- 
cant binding was observed with the mucopolysac- 
charide fraction of connective tissues, but binding 
was found with highly purified acid-extracted 
collagen obtained from several animal sources. 
The optimal pH for binding to purified collagen 
was found to be between pH 3.5 and 4.2. The 
amount of benzoquinoneacetic acid bound to puri- 
fied collagen is proportional to the concentration 
of protein, and about 1 to 2 molecules of the acid 
are bound per molecule of collagen. The behavior 
of the resulting product on carboxymethylcellu- 
lose columns suggest that binding of the acid has 
altered the secondary structure of the collagen 
molecules (a and /? chains). Further experiments 
are in progress to identify the binding sites of 
collagen by separation of the collagen fibrils by 
column chromatography and to degrade the bound 
benzoquine-collagen product enzymatically with 
collagenase. 

4-. Folic Acid and Tyrosine Metolbolism 

During the past few months we have gained 
further insight into the rather complicated mech- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



171 



anism by which folic acid and ascorbic acid inter- 
act to maintain the normal oxidation of tyrosine 
in mamalian liver. We previously had shown that 
the first oxidative step of tyrosine metabolism is 
the oxidation of p-hydroxyphenylpyruvic acid by 
a specific oxidase, and this oxidase is inhibited by 
its substrate in vitamin C-deficient guinea pigs fed 
large amounts of tyrosine, and p-hydroxyphenyl 
metabolities are excreted. If either ascorbic 
acid or folic acid is given to the C-deficient ani- 
mals just before extra tyrosine is fed, inhibition 
of the oxidase is prevented. In vitro experiments 
phenyl metabolites are excreted. If either ascor- 
bic acid or folic acid is given to the C-deficient ani- 
tected the enzyme from inhibition by excess sub- 
strate, but in this system, folic acid was ineffective. 
These results suggested that folic acid must have 
been converted to some active form, possibly a 
reduced folic acid derivative, to protect the oxi- 
dase in view. 

In recent in vitro experiments with p-hydroxy- 
phenylpyruvic acid oxidase preparations from 
guinea pig liver, it has been found that reduced 
folic acid (tetrahydrofolic acid) and some other 
reduced derivatives do protect the oxidase from 
inhibition. Dimethyltetrahydropteridine, dihy- 
drofolic acid and anhydroleucovorin factor are all 
effective, and dimethyltetraptericline is nearly ten 
times as effective as ascorbic acid. Furthermore, 
folic acid (not reduced) and some folic acid antag- 
onists, such as amethopterin, inhibit the oxidase. 
These findings, with our earlier evidence that 
ascorbic acid protects the oxidase by some indirect 
mechanism, suggest that the oxidase has a reduced 
pteridine as a cofactor which can be maintained 
in its active (reduced) form by ascorbic acid. It 
is of interest that a reduced pteridine cofactor is 
known to be required for phenylalanine hydroxyl- 
ase and the reactions catalyzed are similar in that 
p-hydroxyphenylpyruvic acid oxidase also cata- 
lyzes a "hydroxylation" step to form homogentisic 
acid. 

5. Tyrosine Transaminase 

The activity of liver tyrosine transaminase is 
increased up to 8-fold in rats after cortisone ad- 
ministration. In studies on the mechanism of this 
steroid-induced effect, the characteristics of tyro- 
sine-a-ketoglutarate transaminase purified from 
liver of induced and uninduced rats have been 
carefully examined. The enzyme, purified 500- 



fold from both sources, has been found to be much 
less specific than had been previously reported. 
Other aromatic amino acids transaminated by the 
enzyme are: phenylalanine, tryptophan, DOPA, 
/fthienylalanine and some ring-substituted tyro- 
sine analogues. The ratio of tyrosine to trytophan 
transamination activity reaches a constant ratio 
during purification of the enzyme and is not 
changed by various treatments of the purified en- 
zyme. These results would explain the reports 
that "tryptophan" transaminase is also induced 
by cortisone (which was assumed to be a separate 
enzyme). Several derivatives of phenylalanine, 
tyrosine, tryptophan and DOPA were found to 
act as competitive inhibitors of the transaminase. 
This new information will be of considerable value 
in further studies on the mechanisms which regu- 
late the activity of this enzyme in vivo. (Drs. 
LaDu, Zannoni, Seegmiller, Howell, Malawista, 
Goldfinger and N. C. Brown) . 

Studies on Gout 

1. Mechanism of Action, of Colchicine 

Our recent demonstration of an acute goutlike 
inflammatory reaction to crystalline suspensions 
of monosodium urate injected intra-articularly in 
gouty volunteers (1961 annual report) suggested 
the possibility that colchicine might act in termi- 
nating an acute attack of gouty arthritis by sup- 
pressing the inflammatory reaction to sodium 
urate crystals. Experiments both in vitro and 
in vivo were designed to examine this possibility. 

The magnitude of the inflammatory response to 
the injection of a standard dose of a crystalline 
suspension of sodium urate was evaluated before 
and again after the intravenous administration of 
therapeutic doses of colchicine. In addition, the 
synovial effusion was aspirated six hours after 
injection and the leukocyte counts, crystal counts, 
and the percent of crystals undergoing phagocy- 
tosis was determined by direct count in a hemocy- 
tometer chamber. For evaluating the response to 
subcutaneous injection, measurements were made 
of the diameter of the areas of erythema and of 
induration, the warmth (using a thermocouple in 
later tests) and tenderness to pressure. 

In vitro studies : Human leukocytes isolated by 
fibrinogen sedimentation were incubated in plasma 
with sodium urate crystals. C 14 2 is evolved by 
the peroxidative degradation of sodium urate-6- 



172 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



C 14 crystals. The influence on C 14 2 formation 
of colchicine added in vitro or of plasma from 
a patient who had received a therapeutic dose of 
colchicine was determined. Other studies utilized 
a second biochemical parameter of phagocytosis 
first demonstrated by Karnofsky. The increased 
oxidation of glucose-1-C 14 to C 14 2 that occurs in 
response to phagocytosis was determined and the 
effect on C 14 2 formation of colchicine added in 
vitro or in plasma from a patient who had received 
a therapeutic dose of colchicine was evaluated. 

In vivo studies : All studies were performed on 
gouty or non-gouty volunteer patients in the Arth- 
ritis and Rheumatism Branch of NIAMD. Pre- 
treatment of asymptomatic gouty volunteers with 
therapeutic doses of colchicine administered in- 
travenously resulted in a substantial suppression 
in the majority of patients of the clinical evidence 
of an inflammatory reaction to sodium urate crys- 
tals injected intraarticularly or intradermally. 
The suppression was particularly striking in those 
patients who obtained the greatest inflammatory 
response in the absence of colchicine. 

Studies in vitro showed that addition of colchi- 
cine or of plasma from a gouty patient who had 
received a therapeutic dose of colchicine substan- 
tially reduced both biochemical paramters of pha- 
gocytosis. The peroxidative destruction of crys- 
tals of sodium urate-6-C 14 that results from their 
phagocytosis by leukocytes was substantially di- 
minished and the specific stimulation of the oxida- 
tion of glucose-1-C 14 to C 14 2 that accompanies 
phagocytosis was also suppressed by the colchicine 
treatment. 

2. Uricolysis by Human Leukocytes 

Despite the absence of uricase in the human spe- 
cies, one third to one fourth of the uric acid pro- 
duced in the normal human each day is destroyed 
prior to excretion from the body. The prevailing 
view is that the intestinal tract is the sole site of 
such degradation through the action of bacterial 
enzymes. No convincing evidence has previously 
been found of a breakdown of uric acid by any 
human tissues. A peroxidative destruction by 
heme proteins and by verdoperoxidase has been 
demonstrated in vitro by other workers. The 
possibility that a peroxidative destruction of so- 
dium urate crystals might accompany the phago- 
cytosis of urate crystals that is seen in acute gouty 
arthritis led us to seek evidence of such a destruc- 



tion by intact human leukocytes and erythrocytes. 

Human leukocytes isolated by fibrinogen sedi- 
mentation from peripheral blood were incubated 
with plasma containing a suspension of crystalline 
sodium urate-6-C 14 . In duplicate experiments the 
same quantity of sodium urate-6-C 14 in super- 
saturated solution was used. The C 14 2 formed 
by the peroxidative destruction of uric acid to 
allantoin was collected and counted. 

In other studies the leukocytes were sonically 
disrupted and the disappearance of uric acid was 
followed spectrophotometrically by the decrease 
in absorbance at 292 rn.fi. The effect of adding a 
hydrogen peroxide generating system consisting of 
glucose oxidase and glucose was also evaluated. 

Our studies showed that human leukocytes are 
able to break down uric acid. Phagocytosis of 
crystals of sodium urate- 6-C 14 results in a greater 
uricolysis (around 0.3 ^gm/hr/10 6 leukocytes) 
than is observed when the same amount of sodium 
urate is present in solution. Erythrocytes showed 
no significant destruction of sodium urate. De- 
struction of uric acid by sonically disrupted leu- 
kocytes was dependent on a hydrogen peroxide 
generating system and the magnitude of the de- 
struction was comparable to that observed in in- 
tact leukocytes (Drs. Seegmiller, Howell, Mala- 
wista and Klinenberg). 

GASTROENTEROLOGY UNIT 

Whipple's Disease 

Both the clinical and laboratory research activi- 
ties of this group are concerned with problems re- 
lated to intestinal absorption. A major portion 
of the clinical activities has been focused on 
Whipple's disease, a poorly understood disorder 
that produces, as a primary manifestation, a broad 
malabsorption syndrome in association with in- 
filtration of the small-intestine mucosa by muco- 
protein-laden macrophages. We have demon- 
strated in four patients that relapses of this disease 
can be reversed rapidly and extensively by treat- 
ment with adrenoeorticosteroids, together with 
broad-spectrum antibiotics. A fifth patient, 
treated with antibiotics only, also improved, but 
at a much slower rate. All five patients demon- 
strated "bacillary bodies" in their small-intestine 
mucosal biopsies when they were in relapse. With 
remission, these bodies, the nature of which is 
presently obscure, disappeared. After prolonged 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



173 



remissions (IV2 years) attributable to continued 
treatment with adrenocorticosteroids and anti- 
biotics, two patients still show infiltration of the 
intestinal mucosa by mucoprotein-laden macro- 
phages despite their generally satisfactory clinical 
conditions. All five patients presented with hy- 
poalbuminemia when they were in relapse. In 
each case it was possible to demonstrate that ex- 
cessive enteric leakage of albumin was present, 
suggesting that this process is partly, if not 
wholly, responsible for the hypoalbuminemia of 
Whipple's disease. Two biopsies of intestinal 
mucosa known to contain "bacillary bodies" have 
been cultured but no organisms were isolated. 
Studies of relatives of the patients have not as 
yet revealed a familial occurrence of this disease 
in the present series (Dr. Laster). 

Metabolism of D-xylose 

It is common clinical practice to assess intestinal 
absorption in patients by feeding them 25 grams 
of d-xylose and measuring the urinary excretion 
of this pentose during the ensuing five hours. 
Little is known, however, about the pathways of 
metabolism of d-xylose in mamalian tissues, and 
less is known about factors other than intestinal 
absorption which may influence the results of the 
xylose tolerance test. A study is in progress, 
therefore, to delineate the pathways of liver me- 
tabolism of d-xylose in vitro. We have partially 
purified an enzyme activity which appears to cata- 
lyze the dehydrogenation of d-xylose to d-xylonic 
acid using DPN preferentially, but also TPN, as 
the hydrogen acceptor. A number of enzyme ac- 
tivity's properties have been characterized. 

In addition, the oxidation of d-xylose-1-C 14 to 
C 14 2 has been studied in intact normal and thyro- 
toxic guinea pigs. The results suggest that d- 
xylonic acid is an intermediate compound in the 
oxidation, and that thyroxin stimulates not only 
the renal excretion of d-xylose, as others have sug- 
gested, but also the oxidation of the pentose to 
C0 2 (Drs. Laster and Weser) . 

Amino Acid Transport in the Small Intestine 

Because information on amino acid absorption 
in the healthy and diseased human subject is lim- 
ited, we have initiated a study of this physiologi- 
cal process. The first phase of the study has con- 
sisted of an evaluation of active transport of mono- 
aminomonocarboxylic acids by segments of ham- 



ster small intestine. Information so derived will 
now be applied to human subjects. Glycine, L- 
alanine, L-leucine, L-valine and alpha-amino-iso- 
butyric acid are all actively transported in our ex- 
perimental system. The kinetics of their trans- 
port systems conform to Michaelis-Menten anal- 
yses. K t values, analogous to K m values of enzyme 
kinetics, have been derived for each compound. 
Of interest is the finding that an amino acid such 
as glycine may have a greater maximal transport 
rate than L-leucine, but because its apparent affin- 
ity for the transport mechanism is 25-fold less than 
that of L-leucine, glycine is competitively inhib- 
ited in its active transport by L-leucine. Each of 
the five compounds studied was found to be maxi- 
mally absorbed in the lower mid portion of the 
hamster small intestine. Water transport ap- 
peared markedly to influence amino acid transport 
— more rapid water movement producing more 
rapid amino acid transport (Drs. Matthews and 
Laster) . 

Glucose Metabolism by Small-Intestine Mucosa 

The small intestine allegedly derives much of 
its energy for active transport from glucose oxida- 
tion; hence an understanding of this biochemical 
pathway is important in relation to probing mech- 
anisms of the intestine's absorptive functions. In 
addition, pharmacological closes of hydrocortisone 
are known to improve acutely the absorptive capa- 
city of a small intestine involved by nontropical 
sprue without altering the morphological abnor- 
malities which characterize this disease. It is per- 
tinent, therefore, to inquire whether hydrocorti- 
sone influences metabolism by the small intestine. 

Slices of intestinal mucosa from animals or 
from humans metabolize glucose added in vitro. 
Both glucose-1- and glucose-6-C 14 are oxidized to 
C 14 2 , the total conversion being equal to some- 
what less than y 2 percent of the glucose. Hydro- 
cortisone, 10" 3 M, suppresses oxidation of glucose- 
1-C 14 by approximately 20% and of glucose-6-C 14 
by approximately 80%. Similar suppression is 
observed with intestinal mucosa from patients 
with nontropical sprue. Homogenates of animal 
intestinal mucosa have an even lower endogenous 
rate of glucose metabolism, a rate which is affected 
by hydrocortisone in the same maimer as the 
metabolism by slices. The addition of ATP and 
TPN to homogenates in appropriate concentra- 
tions can increase the oxidation of glucose-1-C 14 



174 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



by 20-40-fold but it does not stimulate the oxida- 
tion of glucose-6-C 14 to C 14 2 (it may increase lac- 
tate accumulation, but this remains to be studied) . 
Hydrocortisone will no longer inhibit oxidation 
of glucose-1-C 14 when its rate has been increased 
by ATP and TPN, but it will inhibit the oxidation 
of glucose-6-C 14 in the presence of these cofactors 
(Drs. Laster and Fenster) . 

CLINICAL ENDOCRINOLOGY BRANCH 

The work of the Branch has continued along 
several rather separate avenues of investigation. 
The major areas of emphasis, as in the past, have 
been on the biochemistry of the thyroid gland and 
its hormones and on carbohydrate metabolism. 
Problems in amino acid transport have been given 
additional attention, and an active program deal- 
ing with the physical chemistry of proteins has 
been pursued. An investigation centering on the 
steroid hormones has been initiated. The inter- 
disciplinary character of the staff membership 
continues in evidence. The brief summaries which 
follow demonstrate to some degree the collabora- 
tion and cross-interests of the biochemists, physi- 
cal chemists, organic chemists and clinical inves- 
tigators who comprise the senior membership of 
the staff. This wide spectrum of training, knowl- 
edge and approach to problems has greatly en- 
riched the research program as well as the outlook 
and development of the individual scientists. The 
mutual benefits derived from the presence of visit- 
ing workers from abroad have also been signifi- 
cant. In the past year, the Branch has been host 
to scientists from Japan, India, and Italy, and 
permanent members of the staff have had work 
assignments in France and England. Training 
has been provided for several postdoctoral fel- 
lows and students who have spent varying periods 
of time in the Branch. 

I. Biochemistry of the Thyroid 

A. Iodide Transport 

The ability of various univalent anions to react 
with the iodide transport system of sheep thyroid 
slices has been investigated. Since the 1961 re- 
port, several other ions have been examined by 
measurement of their saturation characteristics. 
The K M and/or K r values ranged from 3-5 x 10 7 M 
to 2 x 10~ 2 M, giving the following series: 

T c O*- < ClOr < ReCv < BF 4 - < SeCN- & S0 3 F- 

<SCN-<I-<N0 3 -<N0 3 -<OCN- sBr 



The partial molal ionic volumes at infinite dilu- 
tion, </> , were determined by pycnometry. With 
the exception of ReCv, S0 3 F~ and SeCN", <£ 
was inversely related to the K values. A linear 
relation between pK and <f> was found between 
the values 25 and 46 cc/mole for <j> . At larger 
volumes, pK declined, but a clear-cut maximum 
was not observed. Except for T c 4 ", which was 
not tested, all of the anions were shown to be 
competitive inhibitors of iodide transport (Dr. 
Wolff and Mr. Maurey). 

The previously described finding that ouabain 
caused inhibition of thyroidal iodide transport 
was investigated in greater detail. Adenosine tri- 
phosphatase (ATPase) activity was measured in 
homogenates and subcellular fractions of thyroid 
and other iodide-transporting tissues of various 
species. A ouabain-sensitive portion of ATPase 
activity paralleled the properties of the iodide 
transport system in the following ways: (1) both 
were comparably sensitive to ouabain over a thou- 
sand-fold range of concentration and in a variety 
of species, (2) they were comparably sensitive to 
six other cardioactive compounds and quinidine 
over a ten thousand-fold concentration range, (3) 
both were half-activated by K + at a concentration 
of 0.9 to 1.4 mM, and partial activation by oua- 
bain was reversed by K + , (4) both were com- 
parably stimulated by thyrotropic hormone. It 
was concluded, therefore, that this Na + — K + re- 
quiring ATPase system is indirectly involved in 
iodide transport (Dr. Wolff and Dr. Halmi). 

The technical matter of the possible interference 
in in vitro iodide transport studies by iodide re- 
leased into the medium from the tissue was studied 
with rabbit thyroid slices which had been "equi- 
librium labeled." The trichloroacetic acid soluble 
material was shown by chromatography and elec- 
trophoresis to be 20 percent in the form of iodide, 
30 percent in peptides, and the remainder in pro- 
tein. It was concluded that the iodide released 
from sheep thyroid slices was insufficient to inter- 
fere with iodide saturation studies (Dr. Wolff). 

Since lecithin derived from the thyroid gland 
had been reported to bind iodide, the behavior of 
other anions was investigated. Partition of radio- 
active ions between aqueous and nonaqueous 
phases was measured at pH 4. Ions which were 
concentrated to a considerable extent by the thy- 
roid (I", ReCv, T c Cv) were bound more strongly 
to thyroidal lecithin than those (Br - , SCN", 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



175 



Mo0 4 =, S0 4 =) for which the thyroid had little or 
no affinity. Binding to the phospholipid is de- 
creased at higher pH, and this pH sensitivity in 
relation to binding of the various ions is under 
study (Dr. Wolff and Dr. Schneider) . 

Thyroxine and I odotyrosine Synthesis 

It has been generally believed that, in the iodin- 
ation of tyrosine, the second iodine atom is intro- 
duced much more readily than the first. Bio- 
logical iodinating system operating in vitro, how- 
ever, usually show monoiodotyrosine (MIT) as 
the principal product. Studies were carried out, 
in collaboration with Dr. M. Berman, OMR, 
NIAMD, and Dr. R. Pitt-Rivers, National Insti- 
tute for Medical Research, Mill Hill, England, on 
the rates of iodination of N-acetyltyrosine and N- 
acetylmonoiodotyrosine by I 2 in aqueous systems. 
At ph 9.0 to 10.5 the rates of introduction of the 
first iodine into the phenyl ring was 5 to 10 times 
faster than the second. The observed reaction 
rates varied inversely with the square of the T 
concentration, in keeping with the following 
scheme of reactions : 



I 2 ^I + + T;I 2 + I<= 
H 2 OPOH 

T + PhenO- 



iI 3 -;I + + H 2 0^±H 2 OI + ; 
?±H 2 + HOI; 
^PhenIO+H + 



The effect of pH on this reaction is now under 
study (Drs. Rail and May berry). 

Further work on model systems for the coupling 
reaction in thyroxine biosynthesis has been carried 
out. The nonenzymatic reaction in which thyrox- 
ine (T 4 ) is formed in 25 percent yield from 4-hy- 
droxy-3,5-diiodophenylpyruvic acid (DIHPPA) 
and diodotyrosine (DIT) was extended to related 
compounds. The coupling of DIHPPA with MIT 
gives 3,3',5'-triiodothyronine in a yield only 
slightly smaller than for T 4 . This method pro- 
vides a practical synthesis for this compound 
which is of biological interest and is known as 
"reverse T 3 ." When DIHPPA is coupled with ty- 
rosine, or when 4-hydroxy-3-iodophenylpyruvic 
acid (MIHPPA) is coupled with DIT, the yields 
are much smaller. The coupling of DIHPPA 
with glycyl-L-DIT-glycine resulted in the forma- 
tion of glycyl-L-T 4 -glycine in 7-9 percent yield. 
This is the first known synthesis of a thyroxyl tri- 
peptide (Drs. Cahnmann and Shiba). 

The mechanism of the reaction of various free 
phenoxy radicals with analogs of tyrosine, and 



with oxygen, were studied. Previous investiga- 
tions showed that analogs of T 4 



R. 

I 



HO 



— 



>— R 2 



Ri 



could be obtained from tri-t-butylphenoxyl 



I 

Ri 



and tyrosine or desaminotyrosme 



( ho ^3 _ei )' 



The effect of electron withdrawing groups at R 3 , 
in place of the electron donating t-butyl group, 
was examined. When R 3 = COCH 3 or COOC 2 H 5 

or CN, the product was 



Ri OH 



<^>-°< 



— Ra. 



Rj 



It appears that these orthophenol ethers are 
formed via unstable ortho-quinol ether intermedi- 
ates. The products of the reaction of the free 
radical with oxygen indicates that unstable hydro- 
peroxides (or peroxides) are the first reaction 
products (Drs. Cahnmann and Shiba). 

In the course of a study of in vitro iodination by 
a thyroid homogenate system, an artifact was 
found to be due to the contamination of commer- 
cial preparations of glucose oxidase with /3-f ructo- 
furanosidase. This resulted in the production of 
peroxide from the sucrose used in preparing the 
homogenate. The enzyme also hydrolized raffinose 
and trehalose, but not maltose, lactose and a-fructo- 
furanoside (Drs. Wolff and Robbins). 

0. lodoproteins 

The physical chemistry of thyroglobulin has re- 
ceived further study. The degrees of unfolding 
and disaggregation of the molecule as a function of 
urea concentration was determined by sedimenta- 
tion, viscosity, optical rotation, ultraviolet differ- 
ence spectra, and tryptophan fluorescence. The 
molecular changes produced by urea were partial- 
ly reversible. 

The effects of iodination of thyroglobulin on its 
molecular configuration has been investigated. 



176 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Only above ~40 percent iodination of the tyrosyl 
groups were configurational changes observed ; at 
higher levels, the protein became denatured. When 
less than 110 moles of iodine were introduced, the 
only important difference between the iodinated 
and the native molecules was the susceptibility of 
the former to thermal or alkaline fragmentation. 
The rate of tryptic hydrolysis was the same for 
native and ligbtly iodinated thyroglobulin (Dr. 
Edelhoch and Mr. Lippoldt) . 

The spectrophotometry titration method, de- 
veloped last year, for the measurement of iodo- 
aminoacids in intact thyroglobulin was applied to 
a study of the iodination of gamma globulin. The 
course of iodination resembled that of thyroglobu- 
lin in certain respects. Unfolding of the molecule 
with urea or guanidine resulted in a "normaliza- 
tion" of the iodination behavior of the tyrosine 
residues, but did not change the production of 
thyroxyl groups, which were formed only at high 
degrees of iodination. Gamma globulin, however, 
was less sensitive than thyroglobulin to urea. The 
similarity between iodination of native gamma 
globulin and thyroglobulin indicates that the latter 
has no special properties in this respect (Dr. Edel- 
hoch and Mr. Schlaff) . 

Most preparations of thyroglobulin are contam- 
inated with a protein having a higher sedimenta- 
tion coefficient ( ~25 S ) . This substance has so far 
not been characterized. By repeated centrifuga- 
tions, and by a sucrose gradient technique, prepara- 
tions enriched up to 70 percent in this component 
have been obtained. The results of spectrophoto- 
metric titration indicate that the 25 S component is 
an iodoprotein which differs in iodoamino acid 
composition from 19 S thyroglobulin. The nature 
of these differences is under investigation (Dr. G. 
Salvatore and Dr. M. Salvatore) . 

D. Thyroxine Transport in Blood 

Studies on the kinetics of the thyroxine-serum 
protein interactions have continued with the col- 
laboration of Dr. Mones Berman (OMK, 
NIAMD) . The rate of quenching of tryptophane 
fluorescence produced by adding thyroxine to the 
protein was measured by means of a rapid-filling 
cell and a fast recorder. A concentration-depend- 
ent quenching rate was observed, and indicated 
that with bovine serum albumin (5.5 x 10~ 7 M, pH 
7.4, room temperature) the binding reaction was 
complete in 100 milliseconds. Although deter- 



mination of the rate constants was not possible 
without further refinements in the instrumenta- 
tion, the great rapidity of the interaction is of 
physiological interest (Drs. Robbins, Rail, and 
Andreoli). 

Work was begun on the isolation of the specific 
thyroxine-binding proteins from human serum. 
This presents great difficulty in the case of the 
alpha globulin (TBG) because of its low concen 
tration (about 1 nig per 100 ml). With Cohn 
Fraction IV-4 as starting material, and using a 
modified chromatographic procedure on diethyl- 
aminoethyl cellulose, it has been possible to obtain 
a TBG fraction contaminated only with one other 
alpha globulin. Efforts to improve the yield and 
purity are continuing. The thyroxine-binding pre 
albumin (TBPA) is also obtained by this pro- 
cedure, and the iron-binding protein, transferrin, 
is also obtained in apparently pure form (Drs. 
Andreoli and Robbins) . 

The interrelationship between thyroxine-bind- 
ing by proteins in blood and in extravascular 
fluids was investigated in mice inoculated with 
Ehrlich ascites tumor. Interesting differences were 
found. Normal serum contained mainly a "post- 
albumin" thyroxine-binding protein, and an alpha 
globulin in trace quantity. Ascites, however, con- 
tained much more of the alpha globulin. In 
tumor-bearing animals, the serum contained in- 
creasing amounts of the alpha globulin, depending 
on duration of disease. The findings suggest the 
possibility that some extracellular thyroxine-bind- 
ing proteins may have an extravascular origin, and 
that some of the serum proteins may be derived 
from the extravascular fluid (Drs. Salvatore and 
Robbins) . 

E. The Chromatographic Separation of Iodoami- 
no Acids 

Studies of the chromatographic behavior of 
iodoamino acids on columns of strong base anion 
exchange resins have been extended. Existing 
methods using aqueous formic or acetic acid have 
been unsatisfactory for the separation of thyroxine 
(T 4 ) and triiodothyronine (T 3 ) . Since this failure 
appears due to strong nonionic binding forces be- 
tween the resin matrix and these compounds, a 
systemic study of the pH dependence of the bind- 
ing in the presence of solvents with high dielectric 
constant and high solvating capacity for iodo- 
thyronine was undertaken. Resin cross-linking 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



177 



and competing anion concentration were also 
varied. It was possible to obtain a high degree of 
differential binding between T 4 and T 3 with two 
resin types of low cross-linkage and with either 
of four solvents. Column chromatography based 
on these findings has resulted in two satisfactory 
techniques for the complete resolution of iodide, 
MIT, DIT, T 4 and T 3 in serum or in thyroid 
hydrolysates (Dr. Lewallen). 

F. Action of Thyroxine on Isolated Systems 

The investigation of thyroxine effects on liver 
mitochondria has continued in collaboration with 
Drs. R. Michel, O. Michel, and S. Varrone at the 
College de France, Paris, France. It has been 
shown for both T 4 and T 3 that the rapid swelling 
induced by these agents was not accompanied by 
detectable metabolism. The binding of these 
compounds to the mitochondria were largely con- 
centration independent over the range 10~" 5 M to 
10" 9 M. The results indicate an absence of ob- 
ligatory coupling between metabolism or binding 
of the hormone and its effect on mitochondria. 

Iodine (I 2 ) (5X10" 6 M) and ICN (10" 6 M) 
have been shown to swell rat liver mitochondria. 
This swelling was reversed by ATP, and prevent- 
ed by albumin, hypertonic sucrose, amytal, anti- 
mycin A, cyanide, EDTA, and dinitrophenol, ir- 
respective of the oxidation-reduction state of the 
respiratory carriers. In all respects, the effects 
of I 2 and ICN mimic that of thyroxine, and the 
possibility is raised that I + may be the effective 
agent in each. I 2 at 4X10 -5 M also caused an 
increase in oxygen consumption of liver mitochon- 
dria and a slight decrease in P/O ratio with suc- 
cinate as substrate. At 4 X 10 -4 M, both respira- 
tion and phosphorylation were suppressed. ICN 
likewise depressed the P/O ratio at 4X10" 5 M, 
but with a decrease in oxygen consumption attrib- 
utable to the cyanide (Dr. Rail). 

G. Studies on Congenital Goiter 

Patients with congenital goiter have been under 
study because they present the opportunity to 
identify specific defects in thyroid function. A 
number of such defects, similar to those described 
by others, have been observed. In the past year, 
however, an unusual defect was found, there being 
one other case in the world literature. In this 7- 
year-old girl with goitrous hypothyroidism, the 
thyroid gland, salivary glands and gastric mucosa 



all failed to concentrate iodide in the normal man- 
ner. After a brief period of treatment with 12 mg 
of KI per day, many of the signs of hypothyroid- 
ism were reversed. Thus, by supplying sufficient 
iodine through simple diffusion, it was demon- 
strated that the subsequent steps in hormone 
synthesis were intact. The role of the iodide 
trapping mechanism in regulation of hormone syn- 
thesis will be clarified by further studies (Drs. 
Robbinsand Wolff). 

II. Physical Chemistry of Proteins 

Emphasis on fluorescence of proteins has contin- 
ued as an extremely sensitive means for detecting 
alterations in molecular configuration. This work 
has been done in collaboration with Dr. R. Steiner, 
Naval Medical Research Institute. The trypto- 
phan fluorescence has been used in conjunction 
with optical rotation difference spectra, and polar- 
ization of fluorescence, to study the influence of 
high pH and urea on the structure of soy bean 
trypsin inhibitor. It was demonstrated that the 
kinetics of the transitions could be followed by 
fluorescence measurements. The trypsin inhibi- 
tor has an extremely stable structure, and is influ- 
enced only at very high urea concentration (9 M) 
at room temperature (Dr. Edelhoch). 

The effect of the solvent on tryptophan fluores- 
cence of protein was investigated, and model tryp- 
tophanyl peptides were employed to evaluate the 
system. The ability of solvents with low dielec- 
tric constants to alter fluorescence intensity ap- 
peared to depend upon the accessibility of the 
tryptophanyl residues to the solvent molecules. 
The accessibility could be varied by various dena- 
turing solvents such as urea, guanidine HCl and 
detergents (Dr. Edelhoch and Mr. Lippoldt). 

The effect of iodination on the structure and 
function of rabbit antibody gamma globulin was 
examined. Detectable changes in protein config- 
uration occurred only when more than 50 moles of 
iodine were introduced. At these levels, aggrega- 
tion occurred. Formation of only 2 moles of MIT 
and 2-3 moles of DIT ( 12.5 moles of iodine) , how- 
ever, were sufficient to reduce the precipitin activ- 
ity of rabbit antithyroglobulin antibody. Activ- 
ity was further reduced at 25 moles of iodine, but 
progressively greater incorporation of iodine had 
no effect. It appears that tryosyl groups which 
are important for antibody activity are preferen- 
tially iodinated. This is in sharp contrast to the 



178 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



lack of effect of iodination on the precipitin ac- 
tivity of the antigen, thyroglobulin (Dr. Edelhoch 
andMr.Schlaff). 

Studies on the genetic coding ratio for protein 
synthesis were carried out in collaboration with 
Dr. M. Nirenberg, LNE, NIAMD, and LCB, 
NHL Molecular weights were determined for 
polyuridylic acid polymers used as "template" 
RNA in an m vitro E. coli ribosomal system, and 
on the C 14 -polyphenylalanine polymers produced. 
The measurements were extremely difficult because 
of solubility problems which required that new 
techniques be devised. The preliminary results 
indicate that the molecular weight ratios are con- 
sistent with a coding ratio near three (Drs. 
Pfuderer and Edelhoch). 

III. Carbohydrate Metabolism 

A. Glucose 

Further progress has been made in elucidating 
the effects of the thyroid stimulating hormone 
(TSH) on glucose oxidation by thyroid tissue. 
By injecting as little as .01 unit of TSH into the 
carotid artery of the dog, stimulation was detected 
within 15 minutes. The effect in vitro on thyroid 
slices was independent of glucose concentration in 
the medium, and so was not primarily due to a 
change in glucose transport. It was not abolished 
by puromycin, which inhibited incorporation of 
C 14 -leucine into protein. Prior incubation with 
nicotinic acid increased glucose-1-C 14 oxidation 
and augmented TSH action. A good correlation 
was found between TPN levels in thyroid slices 
and C 14 2 production from glucose-1-C 14 , indicat- 
ing the importance of this cofactor in glucose oxi- 
dation by the hexose monophosphate pathway. 
Direct measurement of TPN and TPNH showed 
that TSH, acetylcholine, and menadione increase 
the total TPN nucleotides. Epinephrine and 
serotonin also increase glucose oxidation by chang- 
ing the ratio of TPN/TPNH, but do not increase 
the amount of TPN nucleotides (Drs. Field and 
Pa stan). 

In a study of the effect of ACTH on different 
zones of the adrenal cortex, it was shown to in- 
crease phosphorylase activity in the zona fascicu- 
lata but not in the zona glomerulosa. The latter 
was not influenced by angiotensin, which is 
thought to stimulate aldosterone secretion. Dif- 



ferences in glucose uptake and oxidation were also 
demonstrated in the two zones (Drs. Field and 
Williams). 

The mechanism of the hypoglycemia induced by 
ethanol has been investigated further. Poor nu- 
trition was essential for the hypoglycemia, and 
the effect required 44 hours of fasting. Ethanol 
did not inhibit the hyperglycemic effect of glu- 
cagon on the conversion of fructose to glucose, 
but did impair glycogen synthesis from fructose 
and glucose. Ethanol was found, in perfused liver 
experiments, to inhibit gluconeogenesis apparent- 
ly at the step of amino acid deamination. Al- 
though both the decrease in glycogen synthesis 
and the decrease in gluconeogenesis could lead to 
hypoglycemia, the latter effect appeared the more 
important (Dr. Field). 

B. Glycogen Storage Disease 

An assay for debranching enzyme in leukocytes 
was developed and applied to patients with vari- 
ous types of glycogen storage disease. Low values 
were found in one patient who was known to have 
a deficiency of this enzyme in the liver. A study 
of this patient's family revealed values intermedi- 
ate between the normal level and that found in 
the patient, in the mother, father and one sibling. 
Another sibling had a normal value. The de- 
branching enzyme was normal in the leukocytes of 
patients with glycogen storage disease due to glu- 
cose-6-phosphatase and phosphorylase deficiency 
(Drs. Field and Williams). 

G . Tlie mechanism of action of insulin on the liver 

The effect of insulin on the ultrastructure of the 
rat liver has been studied in collaboration with 
Dr. B. Wetzel, LPH, NIAMD, in an attempt to 
localize the sites of intracellular water accumula- 
tion that may accompany the insulin-induced ac- 
cumulation of potassium. Preliminary electron- 
microscopic observations on perfused rat livers 
failed to reveal any volume changes. After 90 
minutes of perfusion with insulin, however, there 
was a decrease in the number of dense bodies 
(lysosomes). There was also a striking change 
in the appearance of the rough endoplasmic re- 
ticulum, similar to changes usually associated with 
increased rates of protein synthesis (Dr. Morti- 
more). 

Further work has been done with the improvec 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



179 



cj^clic perfusion technique which permits the in 
situ establishment of an isolated rat liver circula- 
tion with virtually no interruption of blood flow. 
Insulin concentrations of the order of 10" 10 M 
(6,000 MW) have been shown to have a variety 
of effects: (1) Insulin strongly inhibited the net 
hepatic release of glucose, appearing within 30 
minutes and reaching a maximum of 6-7 mg/g of 
liver in 90-120 minutes. This effect was independ- 
ent of glucose concentration. (2) Insulin in- 
hibited the increase of lactate which normally 
occurs during perfusion. Since lactate is pre- 
sumably derived from glycogen, this effect is 
consistent with a direct inhibitory action of in- 
sulin or glycogenolysis, and suggests that the 
glucose effect is not via insulin action on glucose 
penetration, glucose phosphorylation or glucose- 
6-P hydrolysis, none of which would affect lactate 
formation. (3) Insulin decreased hepatic K + loss, 
the maximum effect occuring by 60 minutes. Since 
the production of an increase of liver K + by the 
isosmotic exchange of NaCl by KC1 in the medium 
also reduced glucose and lactate accumulation, it 
is possible that the carbohydrate effects may be 
secondary to the action of insulin on intracellular 
K + . (4) Insulin inhibited the hepatic release of 
amino acids similar hi time course and degree to 
the previously shown effect on urea. Insulin also 
enhanced the net increase in uptake of amino acids 
which resulted from their addition to the medium. 
Studies are planned to evaluate the possibility that 
this effect results from an influence of insulin on 
protein synthesis or degradation (Drs. Mortimore 
and Mondon). 

D. Galactose and Galactosemia 

The oxidation of C 14 galactose by patients with 
the congenital disease, galactosemia, has been the 
subject of continuing investigation. Three out of 
a total of 10 patients studied were found to 
metabolize 1 gm of intravenously administered 
galactose to a normal extent although they ful- 
filled all the diagnostic criteria of the disease, in- 
cluding a demonstrated enzyme deficiency in their 
erythrocytes. In one subject, several tissues were 
biopsied, and only the liver had the capacity to 
metabolize galactose. 

Further studies were done, in collaboration with 
Dr. Y. Topper, LBM, NIAMD, on the effect of 
progesterone and menthol on galactose oxidation. 
Although previous experiments had shown that 

705-685—63 13 



metabolism of a trace quantity of galactose was 
increased in galactosemics, in the present work no 
effect of the drugs was found when four patients 
were given 1 gm amounts of galactose. 

Experiments designed to study the effect on 
the offspring of galactose intake by pregnant in- 
dividuals were carried out in collaboration with 
Dr. H. Bernstein, O, NINDB. Pregnant rats were 
fed a diet containing 40 percent galactose. Galac- 
tose was found to pass rapidly across the placenta, 
and fetal blood galactose levels were similar to 
those of the mother. The newborn were shown 
to have cataracts, as well as a decrease in liver 
glycogen and phosphorylase, and histological 
changes in renal tubular cells. These studies in- 
dicate that galactosemic infants may suffer damage 
in utero. 

A sensitive and specific spectrophotometry as- 
say for blood galactose was devised, making use 
of the recently discovered enzyme, galactose oxi- 
dase. This method will be useful as a clinical test 
in newborn infants (Drs. Segal and Roth). 

IV. Amino Acid Transport 

Amino acid transport in kidney cortex slices has 
been examined under various conditions, in contin- 
uation of the investigation reported last year. 
Dr. L. Rosenberg, MB, NCI, and Dr. M. Fox, 
MDB, NIAMD, have collaborated in portions of 
this work. 

In human cystinuria, the basic amino acids ly- 
sine, ornithine and arginine also appear in the 
urine. In rat kidney slices, the amino acids ex- 
hibited mutual competitive inhibition but did not 
affect cystine transport. The same was true in 
monkey kidney, but in dog kidney, competition 
between cystine and the dibasic amino acids was 
observed. 

A number of sugars were tested for their effect 
on amino acid transport by kidney slices, but only 
glucose, galactose and fructose produced inhibi- 
tion. In human subjects, urinary excretion of the 
same sugars — in diabetes, galactosemia, and fruc- 
tosuria, respectively — was associated with amino 
aciduria. The intravenous infusion of glucose and 
galactose in normal human subjects has been 
shown to produce an increase in amino acid clear- 
ance by the kidney. 

The movement of sugar into cells is known to 
alter potassium flux, and the dependence of amino 
acid transport on potassium has been postulated. 



180 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Ion effects on amino acid transport were, there- 
fore, studied. Although affected by potassium, 
amino acid transport demonstrated an absolute 
dependence on sodium. Cardiac glycosides have 
been shown to inhibit amino acid transport. 

Salicylate intoxication causes aminoaciduria in 
man. In the in vitro system, as little as 10 mg 
percent of the drug is an effective inhibitor of 
amino acid transport. The mechanism of this 
effect is under investigation. 

Amino acid transport has also been studied in 
thyroid gland slices. There appeared to be two 
different transport phenomena, one for alpha- 
aminoisobutyric acid (AIB) and glycine, the sec- 
ond for other naturally occurring amino acids. 
Pituitary thyrotropic hormone stimulated AIB 
transport, but did not affect the other amino acids. 
No effect on amino acid transport has been ob- 
served from glucose, or drugs such as thiocyanate 
and perchlorate, which influence thyroidal iodide 
transport (Drs. Segal, Roth and Thier). 

CLINICAL HEMATOLOGY BRANCH 

I. Immunologic Studies 

A. Leukocyte Isoantigen Systems 

During the past 10 years many investigators 
have attempted to measure antileukocyte isoanti- 
bodies, but none of the techniques used, which were 
primarily variations of agglutination procedures, 
have been adequate for establishing the specificity 
of leukocyte antigen groups. Complement fixa- 
tion techniques which were developed in the Clini- 
cal Hematology Branch to measure platelet iso- 
antigens and antiplatelet antibodies of clinical 
significance (see reports of previous 2 years) were 
found to be applicable in measuring antileukocyte 
isoantibodies. Use of these complement fixation 
techniques lead not only to identification of specific 
leukocyte antigens, but also to differentiation of 
specific cell lines which contain the antigens. 
Some antigens were found to be shared by granu- 
locytes and lymphocytes, some limited to lympho- 
cytes only, and some shared by granulocytes, lym- 
phocytes and platelets. We have found that anti- 
bodies against these antigens can be used to iden- 
tify specific cell lines through different stages of 
maturity and will no doubt have applications in 
determining developmental origin of different cell 



types. These new antigen systems, which are as 
clearly defined as erythrocyte antigen systems, are 
of considerable interest as genetic markers and no 
doubt of importance in transplantation immunity 
despite their obscurity up to now. 

The following are some of the clearly defined 
antigen groups : PI is the symbol for platelet anti- 
gen, Gr for granulocyte and Ly for lymphocyte. 
The superscript letter defines the locus and the 
number the identified allele. All antigens named 
in the chart have been confirmed by identical anti- 
bodies found in different individuals sensitized by 
transfusion or pregnancy, as indicated. 



Antigen 



1. P1 A1 

2. PlGrLy Bl . 

3. PIGrLy 01 . 

4. Ly D1 

5. PI 

6. PI 

7. PlGrLy_. 

8. Ly 



Antibody 



Post 
trans- 
fusion 



Post 
preg- 
nancy 



Panel 



Num- 
ber 



Percent 
Positive 



306 

230 

80 

96 

127 

165 

150 

40 



98 
37 
30 
31 
7 
99. 5 
70 
65 



(Drs. Shulman, Marder, Aledort) 

B. Significance of Maternal Antibodies Against 
Isoantigens on Leukocytes and Platelets — 
Effects of Infusing These Antibodies 

The fact that platelets and leukocytes have some 
shared antigens has not been known. By anc 
large those groups of investigators who have been 
working on leukocyte antigens have not testec 
platelets and vice versa. One reason for previous 
conclusions that platelets and leukocytes have dif- 
ferent antigens is the fact that maternal antibodies 
produce thrombocytopenia in newborn infants not 
infrequently (see last year's report) but neonatal 
leukopenia is very rare. Nevertheless, some of the 
same antibodies which were found to react with the 
infants' platelets also reacted with leukocytes in 
complement fixation tests, despite the fact that the 
infants were not leukopenic. This discrepancy 
was puzzling until explained by effects of infusing 
maternal antibodies into adults whose cells con- 
tained reactive antigens. Three different isoanti- 
bodies against three different isoantigens sharec 
by platelets and leukocytes were used in these tests. 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



181 



In all instances there was an immediate fall in both 
platelets and leukocytes, but whereas platelets con- 
tinued to fall as antibody was administered at the 
same rate, granulocytes, after reaching a level of 
500 to 1,000 per mm 3 , increased rapidly, sometimes 
to higher than control levels. The transient leuko- 
penia and persistent thrombocytopenia in the face 
of continued administration of antibody produced 
the deceptive picture of specific action against 
platelets. It therefore could be postulated that 
maternal isoantibodies cause neonatal thrombo- 
cytopenia more often than leukopenia because com- 
pensatory mechanisms for maintaining cell levels 
are more effective for leukocytes than for platelets 
(Drs. Shulman, Marder, Aledort) . 

O. Clinical Significance of Isoantibodies Against 
Leukocytes 

During the past five years, various transfusion 
reactions, ranging in severity from mild chills and 
fever to severe and fatal shock, have been blamed 
on isoantibodies against leukocytes. It is gen- 
erally accepted that feibrile transfusion reactions 
which cannot be accounted for by incompatible 
erythrocytes are due to incompatible leukocytes 
given unknowingly. With the identification of 
specific anti-leukocyte antibodies, it was possible 
to evaluate the effects of intentionally mismatch- 
ing leukocytes in transfusions. Such mismatched 
leukocytes, whether given to an individual previ- 
ously sensitized or mismatched by passive infusion 
of antibody into an individual with reactive cells, 
produced a variable clinical response, ranging 
from no reaction whatsoever, to chills and high 
fever. The dose of antigen given and the rate of 
antigen antibody reaction were found to be im- 
portant parameters in determining the severity of 
clinical response. In a number of instances large 
amounts of mismatched leukocytes could be given 
with no effect whatsoever. Further observations 
of this type will help to determine the significance 
of so-called "buffy coat" transfusion inactions 
(Drs. Shulman, Marder, Aledort). 

D. A New Type of Blocking Reaction Used in 
Determining the Presence of Obscure Iso- 
antibodies 

In a number of instances clinically, either as the 
result of a transfusion reaction or failure of trans- 
fused platelets to circulate, isoimmunization 
against platelets or white cells is suspected, but 



cannot be proved because it is impossible to detect 
antibody activity by agglutination techniques or 
even by more sensitive direct complement fixation 
techniques. We have found that some apparently 
nonreactive sera from individuals suspected of be- 
ing immunized are capable of inhibiting comple- 
ment fixation by previously defined antibodies. 
Antiplatelet and antileukocyte antibodies which 
"block" the complement-fixing activity of other 
antibodies against the same antigen are similar 
to the so-called "incomplete" antierythrocyte anti- 
bodies which block the agglutinating activity of 
other antibodies. The newly recognized type of 
blocking antibody can be measured only by their 
ability to interfere with reactions of complement- 
fixing isoantibodies of the same specificity; and 
in order to test for a blocking antibody of this type, 
the corresponding complement-fixing antibody has 
to be available. Blocking antibodies accounted 
for at least half of the antibodies found in mothers 
sensitized by fetal cells and were observed to de- 
velop in transfused individuals. Evidence for 
mixtures of blocking and complement-fixing anti- 
bodies of the same specificity were obtained in sera 
of sensitized mothers and in individuals sensitized 
by transfusion. Blocking antibodies of this type 
probably account for instances when isoimmuniza- 
tion is suspected clinically but antibody cannot be 
detected by agglutination or direct complement 
fixation techniques. 

It has been assumed generally that most anti- 
bodies which cause rapid destruction of cells re- 
quire participation of complement or have strong 
agglutinating capacity. The ability of "block- 
ing" antibodies (which cause neither agglutina- 
tion nor complement fixation) to affect survival 
of cells in vivo was therefore questioned. Block- 
ing antibodies when infused into reactive recip- 
ients were found to produce as rapid and marked 
thrombocytopenia as complement-fixing anti- 
bodies, indicating that blocking antibodies can be 
as effective as complement-fixing antibodies in de- 
pressing some types of cells in vwo. It is ap- 
parent that when more complement-fixing anti- 
bodies are recognized and available, more blocking 
antibodies will be found to account for isoimmuni- 
zation (Drs. Shulman, Marder, Aledort). 

E. Neonatal Thrombocytopenic Purpura 

Last year's report described the techniques de- 
veloped for measuring antibodies in mothers sen- 



182 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sitized by fetal platelets and the clinical course 
of neonatal thrombocytopenic purpura in six chil- 
dren born of four normal mothers. During the 
past year we have had an opportunity to study 
four additional families, as well as three of the 
same mothers studied last year who became preg- 
nant again. In the latter instances we were able 
to predict the occurrence of neonatal thrombocy- 
topenia by a rise in antibody and to prepare for 
post partum treatment of the infant. In all these 
cases that were treated, purpura was much less 
marked than it had been in untreated siblings 
born about 1 year before. This was true despite 
the fact that the antibodies involved were the same 
and were present in higher, if not equal titer. The 
forms of treatment which were found to be effec- 
tive were administration of steroids to the mother 
prior to the birth of the child, and exchange trans- 
fusion of infants. These studies which will have 
to be continued over the years are firmly establish- 
ing the clinical picture of immune neonatal throm- 
bocytopenic purpura, the types of antigens and 
antibodies involved and the best approaches to 
effective therapy (Drs. Shulman and Marder). 

F. Effects of Isoantibodies on Leukemic Cells 

In determining whether isoantibodies against 
specific cell lines would react with cells at differ- 
ent stages of maturity, cells from patients with 
acute and chronic leukemia were compared with 
normal cells. All cells, regardless of their state 
of maturity, contained the antigens found on nor- 
mal cells. Because of this finding, isoantibodies 
were infused into leukemic individuals to measure 
the ability of these antibodies to depress circulat- 
ing cells. Leukemic cells were found to be un- 
usually sensitive to the action of these antibodies. 
It remains to be determined whether this effect 
was due to the greater affinity of leukemic cells for 
antibody, their higher antigen content or their 
limited reserve. The selective action of these iso- 
antibodies against leukemic cells provides an ap- 
proach to the immunologic therapy of leukemia 
and related disorders (Drs. Shulman, Marder, 
Aledort) . 

II. Coagulation Studies 

A. A New Method for Measuring Minimum in 
Vivo Concentrations of Factor VIII Ap- 
plied in Distribution and Survival Studies 
Small amounts of plasma (0.5 to 25.0 ml.) con- 



taining normal Factor VIII concentrations were 
injected into severe hemophiliacs. After a mixing 
period, blood was drawn for 2-stage prothrombin 
consumption determinations. In vivo Factor 
VIII concentrations from 0.02 to 0.8% of normal, 
produced by dilution of Factor VIII in the hemo- 
philiac's plasma, were correlated with percent 
prothrombin consumed (15 to 85%). Standard 
curves prepared in this way were identical in five 
different hemophiliacs, and could be used to meas- 
ure unknown in vivo Factor VIII concentrations 
with approximately ±20% error. This sensitive 
technique permitted measurement of Factor VIII 
survival in vivo for 12 half-lives over a 5-day 
period. Previously available methods were useful 
for, at most, 6 half -lives, covering less than 2 days. 
Prolonged survival measurements gave decay 
curves with two exponential components which 
were not previously discernible. The initial com- 
ponent had a T1/2 of 4 to 5 hours (primarily ex- 
tra vascular diffusion) ; the second component had 
a T1/2 of 9 to 11 hours (degradation and back 
diffusion) . These two components, which did not 
vary with the amount of Factor VIII adminis- 
tered, indicated an extravascular compartment ap- 
proximately 1.5 times the plasma volume. 

These findings are of special importance be- 
cause up to now no distinction has been made be- 
tween the initial phase of extravascular diffusion 
of Factor VIII and the subsequent phase of deg- 
radation and back diffusion. The ability to dis- 
tinguish these two phases by more sensitive meas- 
urements and the constancy of these two phases 
over an extreme dose range permitted description 
of all survival curves by the same two exponents 
as determined by a least square's fit using a digi- 
tal computer program developed by Dr. Mones 
Berman. Knowledge of the size of the extravas- 
cular compartment and the rates of different decay 
phases permit better estimation of Factor VIII 
requirements in therapy. This is particularly im- 
portant now that concentrates of Factor VIII are 
available for treating hemophiliacs (Drs. Shul- 
man and Marder) . 

B. Standardization of Methods of Measuring 
Factor VIII 

In assaying commercial Factor VIII prepara- 
tions, it became evident that agreement between 
laboratories on a standard of potency and on a 
standard method of measuring antihemophiliac 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



183 



factor was hard to obtain. Because of differences 
in methodology and in standards used by Merck 
Sharp and Dohme, the Protein Foundation which 
licenses Fraction I, and our laboratory, an inter- 
est developed on the part of the Division of Bio- 
logics Standards in establishing a standard for 
measuring Factor VIII. This cooperative study 
between the four groups is continuing in an at- 
tempt to prevent unnecessary confusion which will 
arise as Factor VIII concentrates become more 
readily available for clinical use (Drs. Shulman 
and Marder). 

C. Evaluation of an Unusual Form of Painful 
Purpura 

About 7 years ago Gardner and Diamond de- 
scribed the syndrome of autoerythrocyte sensitiza- 
tion which essentially is \h& occurrence of spon- 
taneous painful bruising in females. The 
diagnostic test suggested for this condition was 
injection of the patient's own red cells intraderm- 
ally, a positive reaction being development of a 
painful ecchymosis at the injection site. The dis- 
ease was considered to be caused by sensitization 
against autologous red cells. 

In studying nine patients who developed spon- 
taneous painful bruising, it was found that two of 
these gave no response and one variable response 
to autologous red cells despite the clinical similar- 
ity of hemorrhagic manifestations in all patients. 
Four of the patients responded to injections of 
histamine in trace amounts (2 micrograms) to 
form large painful ecchymoses, and two responded 
to injections of other basic amines and enzymes 
known to release histamine. Others gave no re- 
sponse to histamine or histamine releasers. Study 
of the histamine metabolism of positive reactors 
gave no evidence of abnormality, nor was there 
evidence of abnormality in catecholamine metabo- 
lism. 

Three of the nine patients responded to blood 
transfusions, but it was found in one of these cases 
that an equivalent response was obtained with the 
patient's own blood used in an autologous trans- 
fusion. Only one case has been tested by autolo- 
gous transfusion but there is evidence from other 
procedures used that it is possible to cause fluctua- 
tions and even total regressions of lesions in this 
disease by procedures which contain much in the 
way of psychological suggestion but make little 
sense physiologically. For this reason, a large 



psychiatric component appeared to be present in 
this disorder and Dr. Hart of NIMH is cooperat- 
ing in an attempt to find some psychiatric common 
denominator among these patients (Drs. Shulman 
and Aledort) . 

D. Continued Studies of a Form of Acquired 

Hemophilia Due to an Abnormality in 
Gammaglobulin 

In the well-recognized disorder of acquired 
hemophilia, perfectly normal elderly individuals 
suddenly become hemophiliacs and the gammo- 
globulin fraction of their plasma has the ability to 
inactivate Factor VIII of normal blood. Recent 
articles have appeared in several journals suggest- 
ing that this acquired activity is enzymatic in na- 
ture, but our studies have shown that the kinetic 
characteristics of the reaction are consistent only 
with a bimolecular combination characteristic of 
antigen-antibody reactions. The stoichiometric 
nature of the reaction has been demonstrated both 
in vitro and in vivo. By infusing plasma from a 
patient with acquired hemophilia, transient hemo- 
philia (for periods of several hours) was induced 
in animals and in man. The quantitative rela- 
tionships between the amount of gamma globulin 
and in the amount of Factor VIII inactivated gave 
some indication of the molar concentration of Fac- 
tor VIII in blood and the rate of Factor VIII pro- 
duction in vivo. This indirect approach to meas- 
uring Factor VIII is the only one available at 
present because Factor VIII has not been sepa- 
rated sufficiently from other plasma proteins to 
permit its physical or chemical characterization. 
The reactions studied in acquired hemophilia have 
direct bearing on the problem of autoimmunity. 
Since the apparent antigen is a blood constituent, 
the possible immunologic basis of the disorder is of 
special interest, for current concepts of autoim- 
munity preclude autologous circulating blood con- 
stituents as possible agents of sensitization (Drs. 
Shulman and Marder) . 

E. Continued Studies of the Significance of 

Changes in Fibrinogen Levels in Familial 
Mediterranean Fever 

In association with Dr. Wolff of NIAID, we 
have continued measuring the fluctuations in 
fibrinogen which occur in patients with familial 
Mediterranean fever. These patients have peculiar 
recurrent episodes of fever associated with a van- 



184 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ety of abdominal and other systemic symptoms. 
The patterns of periodic change in fibrinogen 
which have been measured are comparable to 
changes in fibrinogen associated with pyrogenic re- 
actions or the generalized Schwartzman reaction. 
Since the etiology of the disorder is still quite 
obscure, it is hoped that continued studies on more 
individuals will document changes in fibrinogen 
as an integral part of the disease and thus provide 
a more definite lead in unravelling the patho- 
genesis of the disorder (Drs. Shulman and Wolff) . 

PEDIATRIC METABOLISM BRANCH 

Cystic fibrosis of the pancreas and diseases of 
glycogen storage in their various biochemical, 
physiologic and clinical manifestations have been 
the object of continued investigation in the year 
1962 in the Pediatric Metabolism Branch. The 
program of diagnostic studies on the celiac syn- 
drome and other malabsorptive states initiated in 
the past years was brought to a conclusion. 

Cystic Fibrosis of the Pancreas 

This generalized disorder of children, adoles- 
cents and young adults is thought to be caused by 
an inborn error of metabolism as yet undefined. 
The basic defect, whatever its nature, causes a 
widespread disturbance in exocrine gland function 
and leads to a variety of manifestations due to ab- 
normal physicochemical behavior of mucous secre- 
tions and a unique electrolyte abnormality of 
sweat. The link between the electrolyte and mucus 
abnormality is unknown. 

Investigations have been directed to elucidation 
of the pathogenesis of this disease. 

Biochemical and Immunological Studies of 
Macromolecules in Normal Controls and 
Patients With Cystic Fibrosis of the 
Pancreas 

In an attempt to define the basic defect in cystic 
fibrosis of the pancreas, a coordinated effort was 
planned using both biochemical and immunologic 
methods. In the first phase of the investigation, 
the location of an abnormal constituent common to 
all organs and tissues in fibrocystic patients is 
being sought by Immunoelectrophoresis as a screen- 
ing technique. Immune sera have been produced 
in rabbits by immunization with macromolecular 
antigens recovered from urine of patients with 



cystic fibrosis and of normal controls (Drs. Gab- 
riel, Pallavicini, Raunio, Talamo, di Sant'Agnese, 
Lietman, and with the cooperation of Dr. Seymour 
P. Halbert, Columbia University, New York). 

Chemical Investigation of Glycoproteins in 
Sweat of Patients With Cystic Fibrosis and 
Normal Controls 

The finding of elevated sweat electrolyte levels 
in patients with cystic fibrosis prompted an in- 
vestigation for carbohydrate-containing macro- 
molecules in this secretion. 

Sweat was collected from normal controls as 
well as from patients with cystic fibrosis by ther- 
mal stimulation in a constant-temperature room 
and frozen immediately. The analysis revealed 
the presence of precipitable, nondialyzable, poly- 
saccharide-protein complexes containing galac- 
tose, mannose, fucose, N-acetylglucosamine, N- 
acetylgalactosamine and N-acetylneuraminic acid. 

Paper chromatography as Avell as analytical 
data suggested that regardless of age the subjects 
tested could be divided into three different groups, 
according to the following ratios of total hexose 
to fucose: 30, 12 and 4. All patients with cystic 
fibrosis were in the last group. As a similar ab- 
normality with an increase in fucose and a de- 
crease in sialic acid was found in previous studies 
in duodenal contents of fibrocystic patients, this 
appears to be a general property of individuals 
with this disease and thus gives rise to important 
pathogenetic consideration. 

This investigation has demonstrated for the first 
time the presence of neutral heteropolysaccharides 
in normal and pathologic human sweat. It has 
also reaffirmed the inverse relationship of fucose 
to sialic acid under normal and pathologic condi- 
tions which appears to be a general biologic find- 
ing (Drs. Pallavicini, Gabriel, di Sant'Agnese and 
with the cooperation of Drs. Whedon and 
Buskirk) . 

Biosynthesis of Mucoproteins 

The first phase of the investigation in collab- 
oration with Dr. G. Ashwell was completed with 
the isolation of a new precursor in the biosyn- 
thetic pathway of mucopolysacchrides. This com- 
pound isolated from hen oviduct was identified as 
being uridine-diphosphate-N-acetylglucosamine- 
6-phospho-l-galactose (Dr. Gabriel). 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



185 



Glycogen Storage Disease 

Systematic clinical and chemical studies were 
continued of the diseases of glycogen storage, a 
group of disorders due to errors of carbohydrate 
metabolism leading to accumulation of this poly- 
saccharide in various organs and tissues of the 
body. 

Work is now in progress to make available to us 
all chemical systems necessary to describe fully 
the various disorders in terms of function of en- 
zymes involved in glycogen metabolism under 
physiologic and pathologic conditions. 

In the past year study of the phosphorylated 
intermediary compounds of carbohydrate metabo- 
lism was continued and extended to most of the 
known types of glycogen storage disease. Con- 
trary to accepted opinion it was conclusively 
shown that such compounds are not increased in 
the various blood fractions of patients with this 
disorder. 

Investigations have been performed on tissues 
obtained on surgical biopsy, as well as at necropsy, 
in three patients with Pompe's disease, a fatal 
form of generalized glycogen storage. The ab- 
sence of maltase in this type of disorder was con- 
firmed by means of a sensitive assay developed in 
this laboratory. It was shown that this enzyme, 
formerly not thought to play an important role in 
glycogen metabolism, resides in the soluble frac- 
tion of tissue homogenates, thus allowing its fur- 
ther purification and characterization. Experi- 
mental evidence has been obtained that there are 
several proteins exhibiting maltase activity and 
it is planned to study this further by the applica- 
tion of immunologic techniques (Drs. Gabriel, 
Lietman, di Sant'Agnese and Powell). 

Intestinal Malabsorption in Children 

Two diagnostic tests for malabsorption have 
been refined and simplified and their reliability 
has been evaluated. They will be reported in the 
literature shortly. It is expected that they will be 
of practical value to pediatricians confronted with 
patients with malabsorption. 

Deficient fat absorption is common denominator 
to the many diseases which cause intestinal mal- 
absorption, but its detection has been hampered by 
the lack of simple and reliable tests for its pres- 
ence. During the past year, we have revised and 
simplified a method in which lipiodol is fed to the 



patient and the subsequent urinary excretion of 
the iodine released from the absorbed lipiodol is 
measured by a semiquantitative tube dilution 
method. The results obtained with this test were 
compared with the results of determinations of 
4-day fat balances in patients with steatorrhea due 
to various causes and in control subjects. The 
lipiodol test was positive in those children with 
steatorrhea and negative in those without steator- 
rhea, indicating that the revised lipiodol test is a 
simple and reliable screening test for steatorrhea. 
Although the assessment of a patient's ability to 
absorb the pentose, cl-xylose (xylose tolerance test) 
has been found to be extremely useful in screening 
adults for malabsorption and to surpass in clinical 
value the glucose tolerance test, the xylose toler- 
ance test has not yet been applied to the pediatric 
age group. Accurate data have, therefore, been 
collected for normal children and for children 
with various pathologic conditions. Evidence has 
been gathered to show that the xylose tolerance 
test is more useful than the older glucose tolerance 
test. Xylose absorption appears to be impaired 
primarily in diseases leading to altered fat and 
carbohydrate absorption from the small intestine 
(Drs. Jones and di SantAgnese). 

METABOLIC DISEASES BRANCH 

Mineral Metabolism Studies 

/. Significance of Various Nutritional Factors 
Including Dietary Calcium Intake in Osteo- 
porosis 

Continuing metabolic studies from this Branch 
further substantiate the significant relationship 
between dietary calcium intake and the pathogen- 
esis and possible therapy of postmenopausal and 
senile osteoporosis, a disorder resulting in thinning 
bones more susceptible to fracture. Until the 
work of this Branch and that of two British labo- 
ratories was reported, the accepted concept of the 
pathogenesis of this disorder held that it was the 
result solely of impaired bone matrix formation 
due to hormonal imbalance. The hypothesis of 
multiple etiologic factors affecting mineral utiliza- 
tion has now become more widely accepted. 

A. Effect of Increasing Dietary Intake of 
Calcium on Calcium Balance. To the present 
time, fourteen patients with postmenopausal, se- 
nile, or idiopathic osteoporosis have been studied 






186 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



at several different calcium intake levels from 150 
mgm. to 2.4 grams per day, with balance studies 
of calcium, phosphorus, and nitrogen carried out 
for at least 30 days at each intake level. Three 
types of response have been noted : (a) one group 
had low customary intakes of calcium (less than 
300 mgm. per day) on which they were in negative 
calcium balance ; with increasing dietary calcium, 
increased amounts of calcium were retained, net 
storage of mineral appearing at about 800 to 1000 
mgm. per day of intake (four patients) ; (b) a 
second group had relatively normal or even high 
customary intakes of calcium (over 600 mgm. per 
day) on which they were in negative calcium bal- 
ance ; as dietary intake was increased further, cal- 
cium balances became less negative, with net stor- 
age of mineral appearing at intakes over 1,600 
mgm. per day (four patients) ; (c) the third 
group was similar to the first in that customary 
calcium intake was low. Increasing the dietary 
calcium to 800-1600 mgm. per day resulted in net 
retention of mineral; at higher intakes, however, 
negative balance again resulted (three patients), a 
significant finding which requires further study. 
The data obtained with two additional patients 
have not as yet been evaluated. Two patients who 
demonstrated storage of calcium at high intake 
levels have been reevaluated after more than a 
a year of high dietary calcium and continue to 
demonstrate positive balances, although to a lesser 
extent, indicating development of some degree of 
adaptation to elevated intakes. 

B. Effect of Dietary Phosphate on Mineral 
Retention. The previously noted relationship 
between dietary calcium and phosphate for opti- 
mal absorption and utilization of these two 
elements is being investigated further by manipu- 
lation of both absolute amounts and relative pro- 
portions of these nutrients in the diets of two 
patients with osteoporosis. In addition, the role 
of dietary phosphate has been explored in a patient 
with "phosphate diabetes" (de Toni-Fanconi syn- 
drome) and in a patient with Vitamin D resistant 
rickets. In both patients, evidence for mineral 
retention and bone formation has been obtained 
in the absence of therapeutic dosages of vitamin 
D, by increasing dietary phosphate alone. These 
observations suggest that dietary phosphate and 
mechanisms for phosphate absorption are of sig- 
nificance in the retention of bone mineral. 



C. Effect of Dietary Fat on Mineral Reten- 
tion. Studies by this Branch had previously 
noted that occult chemical steatorrhea was present 
in the patients with osteoporosis who failed to store 
calcium on high intakes, with no other evidence 
(intestinal biopsy, xylose tolerance) for malab- 
sorption. Dietary fat has been varied at two die- 
tary levels of calcium in three patients with osteo- 
porosis. Preliminary results indicate that at high 
calcium intakes, 100 gm. of dietary fat per day 
results in chemical steatorrhea and decreased cal- 
cium absorption as compared with 20 gm. of die- 
tary fat per day. At moderate calcium intakes, 
calcium absorption is greater at 100 gm. of fat 
per day than at the lower intake of fat, and no 
steatorrhea is seen (Drs. Whedon, Lutwak, Laster, 
Gitelman, Fox, and Shapiro) . 

II. Studies of Mechanisms of Nutritional and 
Hormonal Influence on Bone Metabolism, 

Radioisotopic measurements of calcium kinetics 
in association with metabolic balance studies con- 
tinue to be made for estimations of pool size and 
rates of deposition of mineral into bone via intra- 
venous administration of the isotope. In addition, 
isotope has been administered orally to measure 
directly the degree of absorption of calcium from 
the diet. Tracer studies have also been carried out 
with in vitro systems in attempts to arrive at 
mechanisms of calcium transport and retention. 

A. Intravenous Isotope Studies. Results of 
34 tracer studies in patients with osteoporosis con- 
firm the finding first reported by this group that 
the rate of calcium incorporation into bone is 
normal in this disorder. The increased retention 
of calcium observed by balance measurements with 
increasing dietary intake of this element is not 
associated with any increase in the bone incorpora- 
tion of calcium, suggesting that the mechanism of 
retention is by decreased bone resorption. 

Administration of corticosteroids results in de- 
crease in bone formation, as well as in bone resorp- 
tion. Formation is decreased to a greater extent 
than is resorption in patients who develop nega- 
tive calcium balance. 

The conclusions regarding bone formation and 
resorption arrived at from isotopic tracer studies 
have been confirmed by microradiographic ex- 
amination (by Dr. Jenifer Jowsey of Albert Ein- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



187 



stein Hospital, Philadelphia, Pa. ) of bone biopsy 
specimens from these patients. 

B. Oral isotope Studies. Both the rate and 
amount of calcium absorbed from the gastrointes- 
tinal tract have been estimated from absorption 
and excretion of orally administered calcium-45 
in two patients with osteoporosis and two patients 
with idiopathic sprue. These studies will be re- 
peated in both groups after appropriate treatment. 
Preliminary observation of less than 1% absorp- 
tion of isotope in the sprue patients explains the 
finding of hypocalcemia in this frank malabsorp- 
tion syndrome (Drs. Lutwak, Whedon, Gitelman, 
Fox, and Shapiro). 

C. Phosphoethanolamine Metabolism in 
Bone Disease. It has been suggested that phos- 
phoethanolamine (PET), a naturally occurring 
phosphorylated amine, may act as an inhibitor of 
mineralization of bone. A new sensitive, automat- 
ic technique for analysis of this amine was de- 
veloped. Previous results from other laboratories 
had indicated that this substance is absent from 
the urine and serum of normal individuals, and 
that its presence signified the diagnosis of hypo- 
phosphatasia. By the use of the devised tech- 
niques it has been shown that PET is present in the 
serum and urine of all individuals. The rate of 
disappearance of infused PET from the serum 
was found to be directly related to the serum alka- 
line phosphatase level. Preliminary compartmen- 
tal analysis of the disappearance curves indicate 
the presence of a renal threshold for this amine 
(Drs. Gitelman and Lutwak). 

D. The Role of Dermal Losses in Calcium 
Balance. A new technique for continuous col- 
lection of insensible perspiration losses of mineral 
was developed and applied to patients with osteo- 
porosis, as well as to young normal females. Un- 
der conditions of moderate, nonsweating activity, 
loss of calcium, phosphorus, and magnesium from 
the skin was found to be less than 10 mgm. per 
day of each, an amount which therefore may be 
disregarded in calculations of metabolic balances 
conducted under these conditions (Drs. Gitelman, 
Lutwak, and Whedon). 

E. Factors Affecting Renal Excretion of 
Phosphate, Glucose, and Amino Acids. A pa- 
tient with seA^ere osteomalacia and excessive renal 
losses of phosphate, amino acids, and glucose (de 
Toni-Fanconi syndrome) has been studied on 
metabolic balances at varying levels of calcium 



and phosphate intake. Prolonged induced hyper- 
calcemia reduced the rate of phosphate excretion, 
but not to normal levels, without affecting the 
glycosuria or amino aciduria. In normal control 
subjects, infusions of glucose or galactose resulted 
in diminished reabsorption of phosphate and 
amino acids. Explanation for these findings is 
being sought via related in vitro studies (Drs. Fox, 
Gitelman, Lutwak and Whedon, in collaboration 
with Drs. Segal and Thier of the Clinical Endo- 
crinology Branch, NIAMD, and Dr. Rosenberg 
of NCI). 

F. In Vitro Studies of Amino Acid and Phos- 
phate Renal Transport. Glucose, galactose and 
fructose diminished amino acid transport in rat 
kidney cortex slices studied in vitro. The ionic 
composition of the medium for optimum transport 
has been defined. In similar systems, the transport 
of inorganic phosphate has been studied, demon- 
strating an accumulation of P-32 against a con- 
centration gradient. These studies are being cor- 
related with the in vivo observations described 
above (Dr. Fox, in collaboration with Drs. Segal 
and Thier of CEB, NIAMD, and Dr. Rosenberg 
of NCI). 

G. In Vitro Studies of Calcium Uptake by 
Skin. An in vitro system has been developed, 
demonstrating for the first time uptake of calcium- 
45 by surviving slices of rat epidermis. This up- 
take is inhibited by dinitrophenol, and is, presuma- 
bly, under metabolic control. It is planned to ex- 
tend these studies to slices of human skin obtained 
from patients with different diseases of calcium 
metabolism (Drs. Shapiro and Lutwak) . 

H. Magnesium Metabolism in Bone Disease. 
Using a new automatic technique for the estima- 
tion of magnesium, metabolic balances of this 
cation are being determined in patients with osteo- 
porosis and related bone diseases, in an attempt to 
define the interrelationships of magnesium with 
calcium and phosphate metabolism. Preliminary 
results demonstrate that the dietary requirement 
for balance of magnesium is quite variable from 
patient to patient and increases with increasing 
dietary calcium (Drs. Lutwak and Gitelman). 

I. Calcium-Binding by Serum Proteins. It 
was observed repeatedly during the first 24 to 48 
hours after intravenous Ca-45 administration that 
the specific activity of the serum calcium was con- 
sistently higher than that of the urinary calcium. 
In vitro studies using ultracentrifugation and ul- 



188 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



trafiltration have shown that the specific activity 
of the protein-bound calcium is higher than that 
of free calcium in serum incubated with tracer. 
These results suggest that kinetic studies of calci- 
um metabolism based on isotopically derived data 
may be in modest but significant error due to mass 
differences of the radioactive and stable isotopes 
(Dr. Lutwak). 

J. Interrelationships or Dietary and Fecal 
Calcium and Fat in Rats. Rats were placed on 
diets containing no, moderate, or high amounts of 
calcium, labelled with Ca-45 and no fat, fatty acid, 
or triglyceride fat of the myristic or palmitic 
family, labelled with C-14. The feces were col- 
lected for 6 days and analyzed for calcium, free 
fatty acids, soaps, and triglycerides, as well as for 
their specific activities. Evidence has been ob- 
tained for a recycling mechanism for fat excretion. 
In addition, the interrelationships of fat and cal- 
cium excretion were found to be dependent not 
only upon the total amount of fat in the diet, but 
also upon the chemical nature of the fat (i.e., free 
or glyceride) and the length of the carbon chain 
(Drs. Werner and Lutwak) . 
K. Iodine Balance in Normal Subjects. Iodine 
balance studies were carried out at several levels of 
dietary iodine intake in six normal subjects, com- 
paring the excretion of iodide in fractional col- 
lections of urine and stools with total 24 hour ex- 
cretion, as well as with serum inorganic iodide and 
protein-bound iodide levels and with salivary io- 
dide concentrations. It was concluded that uri- 
nary iodide/creatinine and fecal iodide/nitrogen 
ratios provide adequate bases for estimates of 24- 
hour excretions for large scale surveys of popula- 
tions. Salivary and serum iodide levels were ap- 
proximately related to dietary intakes (Drs. Lut- 
wak and Whedon, in collaboration with Drs. 
Vought, London, and Dublin of the Epidemiology 
Branch, NI AMD). 

Energy Metabolism Studies 

Studies of human energy metabolism involving 
use of the Metabolic Chamber for continuous, 
long-term analysis of respiratory oxygen and car- 
bon dioxide exchange are concerned with a num- 
ber of physiological and metabolic problems. 
These investigations deal with disordered metab- 
olism in obesity, effects of various agents on fat 
mobilization and oxidation, mechanisms of tem- 
perature regulation as affected by hormonal ac- 



tion and in various diseases, and the action of digi- 
talis. Collaboration with increasing numbers of 
groups in this and in other institutes is now a 
prominent characteristic of the activities of the 
Metabolic Chamber staff. 

/. Physiological Studies of Obesity 

Although no one has seriously challenged the 
principle that obesity develops as the result of a 
continuing intake of calories which is in excess of 
those needed to provide energy for the various 
processes and activities of the body, for some time 
questions have been raised concerning possible ab- 
normalities in obese individuals in the rate of ca- 
loric expenditure under various conditions and in 
the processes of synthesis, mobilization and oxida- 
tion of fat. In essence to date, varying abnormal- 
ities have been noted in these studies and by other 
workers which suggest that extensive further 
work will be necessary under carefully controlled 
conditions to sort out what appear to be numerous 
different ways in which fat metabolism may be 
disturbed in the obese — at various stages and de- 
grees of obesity, at various ages, and under the in- 
fluence of numerous environmental (and possibly 
genetic) factors. To date the biophysical factors 
thus far noted (by this group) which favor con- 
servation of calories in the obese as compared to 
normal individuals (diminished physical activity 
and less caloric expenditure on exposure to cold) 
have been much more consistent than the meta- 
bolic, and with respect to the latter certain observa- 
tions reported by others have not been confirmed. 

A. Paired Studies of Energy Expenditure. 
Since the Metabolic Chamber analytic apparatus 
presents an unique system for precise measure- 
ment of energy expenditure under a full range 
of physical activities, an effort was made to deter- 
mine the presence or absence of quantitative dif- 
ferences between obese and lean in energy expen- 
diture over an extended period. Two young men 
were studied who were matched in age, height and 
weight, the only evident difference being that one 
had been formerly obese and the other never obese 
in his lifetime ; for a 3 months' period they lived 
together and followed an identical caloric intake 
and physical activity protocol. Following a 1 
month period of weight maintenance, a 6 week 
period of over-nutrition was instituted followed 
by a 2V2-week period of undernutrition to bring 
the subjects back to starting weight. During over- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



189 



nutrition the formerly obese subject clearly out- 
gained the lean subject, the difference being ac- 
counted for by a lesser increase in energy expen- 
diture as determined from periodic Chamber ex- 
periments lasting many hours. Differences in 
body composition were obscured by the experi- 
mental error of these measurements, although it 
appeared that approximately 75% of the weight 
gain by each subject was fat. The meaningfulness 
of such difficult paired experiments is uncertain, 
but it seems important to make further effort by 
similar careful measurements to detect, if present, 
differences in caloric turnover in relation to devel- 
opment of obesity in man. 

B. Metabolic Studies of Complete Fasting. 
Only preliminary data are available on the effects 
of an 18-day complete fast in a 24-year-old 150 kg. 
obese male (45% fat). Unusual features were no 
change in resting energy expenditure for first 5 
days of the fast (in previous caloric reduction 
studies reduction in resting energy expenditure 
began promptly) and development of definite pro- 
longed ketosis (other investigators have reported 
"resistance to ketosis" in obese subjects during 
periods of fasting) . Preliminary blood lipid data 
suggested ready mobilization of fat in this subject 
during the initial five days of fasting, then dimin- 
ished mobilization of lipid stores (Drs. Buskirk, 
Thompson, Lutwak and Wheclon). 

77. Effects of Various Pharmacologic Agents on 
Fat Metabolism and Metabolic Rate 

Various agents known to alter either the rate 
of mobilization or oxidation of free fatty acids 
(FFA) were given to either normal or hyperthy- 
roid patients in an attempt to alter FFA turnover 
and determine the relationship between metabolic 
rate and FFA turnover change. Infusion of glu- 
cose was found to depress FFA but elevate oxygen 
consumption presumably through the specific dy- 
namic effect of glucose. Suxamethonium and 
arfonad (ganglionic blocking agents) were then 
tried because these two drugs are known to depress 
the mobilization of FFA ; initial experimentation 
was unsuccessful because increased heat loss from 
the body surface promoted shivering and a resul- 
tant, elevated metabolic rate. When the subjects 
were covered with blankets to curtail heat loss, a 
parallelism between the decrease in FFA turnover 
and metabolic rate was observed. In more recent 



studies, FFA turnover was increased with nor- 
epinephrine infusion and metabolic rate was 
simultaneously elevated. When the increased 
mobilization of FFA after nor-epinephrine was 
blocked with pronethelon, the usually observed 
increase in metabolic rate after nor-epinephrine 
was also blocked. Although minor interindivid- 
ual variation in these several responses has been 
observed, it appears that resting metabolism can 
be measurably altered by induced changes in free 
fatty acid turnover (Drs. Steinberg and Nestel, 
NHI, and Buskirk and Thompson, NT AND). 

///. Physiologic Studies of Temperature Regula- 
tion 

A. Effect of Triiodothyronine Administra- 
tion. As a means of obtaining fundamental phys- 
iological information on the interplay of central 
and peripheral temperature regulation mecha- 
nisms, experiments before and after triiodothyro- 
nine administration were undertaken to study tem- 
perature regulation in the cold under conditions 
of elevated heat production and heat loss as pro- 
duced by an induced hyperthyroid state. 

Three subjects initially were exposed to cold air 
10° C. (50°F) for 2 hours to secure control ob- 
servations. Triiodothyronine (T 3 ) was then giv- 
en orally for 2 weeks in gradually increased dosage 
until a level of 350-400 ^.gm per day was attained, 
and cold exposures were repeated. Compared to 
control cold exposures, the elevation in body core, 
tympanic membrane, esophageal and rectal tem- 
peratures which took place in the control state 
failed to take place after T 3 . Since vasoconstric- 
tion was less marked and mean weighted skin tem- 
perature remained at a higher value when on T 3 , 
heat loss was greater ; these differences in response 
to cold in body temperatures and heat exchange 
were consistent in all three subjects. The heat 
production pattern (relative change in oxygen 
consumption) in the cold was not remarkably 
altered by T 3 from control observations. Resting 
heat production prior to cold exposure was ele- 
vated by T 3 in two and unchanged in one of the 
three subjects. Time to onset of shivering was de- 
the same following T 3 as it was in tests before the 
layed in one but unchanged in two subjects. After 
shivering started, heat production response was 
the same following T 3 as it was in tests before the 
hormone was given; heat production during the 



190 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



final hour in the cold was unchanged in one and 
elevated in two subjects (approximately 10 and 
40kcal/(m 2 .hr)). 

If a heat maintenance center is located in the 
posterior hypothalamus, and it is sensitive to 
direct temperature stimulation but relays cold re- 
ceptor initiated impulses from the periphery 
(scheme of Benzinger), there should be a delay 
in shivering and a diminution in heat production 
in the cold during the period of administration 
of T 3 . If the anterior hypothalamus functions as 
a direct sensor and inhibits the posterior center 
less and less as it is cooled, then heat production 
in the cold should be elevated more after T 3 than 
during control cold exposures. Actually, the ex- 
perimental data suggest that both schemes of tem- 
perature regulation are operative. As measured, 
the relative increase in heat production in the cold 
was not dramatically altered after T 3 , which may 
indicate, if the regulation schemes are correct, that 
in the hyperthyroid state a relatively smaller stim- 
ulus is initiated from the body surface and a lesser 
inhibition occurs of the posterior by the anterior 
hypothalamus. The net heat production follow- 
ing T 3 would therefore be no different from nor- 
mal, as observed, because of compensation of one 
by the other of these mechanisms (Drs. Buskirk 
and Thompson, NIAMD, and Nestel and Stein- 
berg, NHI). 

B. Thermoregulation Against Cold in Nor- 
mal Subjects and in Patients with Recurrent 
Fever or Experimental Malaria. Investigations 
of thermoregulatory responses to cold air are be- 
ing continued to extend background information 
for similar studies on patients with various dis- 
eases. As described in earlier reports, the insulat- 
ing effect of subcutaneous body fat and total body 
fat in moderating the impact of cold on the body 
has been partially but not completely character- 
ized. It is apparent, however, that body fatness 
must be taken into account to assess properly the 
thermoregulatory responses in patients. Eleven 
patients with recurrent fever and four with ex- 
perimentally induced malaria have been exposed 
to cold air (50°F) each on two or more occasions. 
Although the thermoregulatory responses fre- 
quently fell within the normal range established 
by earlier Metabolic Chamber work, the follow- 
ing findings suggest possible abnormalities in the 
reaction to cold in patients with recurrent fever : 
(1) Heat production was frequently great- 



er than would have been predicted from 
studies on normal subjects of comparable 
body fatness. 

(2) Deep body temperatures (ear, esopha- 
geal or rectal) frequently fell immediately and 
more rapidly following exposure to cold in 
contrast to the early elevation (0.1-0.3°C) 
in core temperature usually seen in normals 
(indicating a defect in heat conservation phys- 
iological mechanisms) . 

(3) Intraindividual variation in heat pro- 
duction and heat loss appeared to be more 
marked in patients with recurrent fever than 
in normals. The final response appeared to 
be at least partially geared to the febrile- 
afebrile periodicity. 

(4) Cold exposure following experimental 
malaria induced, in one patient, an elevated 
heat production, greater peripheral heat loss, 
and more rapid core cooling than seen in con- 
trol experiments performed in this man after 

administration of malarial parasites but be 
fore malarial chills and fever became evident. 

These studies indicate that long-standing recur- 
rent fever and experimental malaria may, in some 
patients, disturb temperature regulation against 
cold to a measurable extent and that further ex- 
ploration of the nature and extent of these ab- 
normalities would be worthwhile, as potentially 
applicable to improved therapeutic management 
of febrile illnesses (Drs. Thompson and Buskirk, 
NIAMID, and S. M. Wolff, NIAID). 

C. Response to Heat and Cold Exposure in 
Patients With Hansen's Disease (Leprosy). 

Studies of thermoregulatory responses which 
may be ultimately significant for various derma- 
tologic diseases involving extensive areas of the 
skin have been initiated in Hansen's disease in 
collaboration with NIAID. Three patients with 
skin and subcutaneous lesions involving approxi- 
mately one-fourth to one-third of their body sur- 
face were exposed to: (a) 15.5°C (60°F) air 
for 2 hours, and (b) 37.7°C (100°F) (40-50% 
K.H.) air for 1 hour. The patients rested in the 
supine position in the cold and sat or exercised on 
the treadmill (3 mph. zero grade) in the heat. 

Heat production, as measured by oxygen con- 
sumption, in the cold was elevated in all three sub- 
jects over values commonly observed for normal 
subjects. In two patients body core cooling started 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



191 



immediately following exposure to cold, (as seen 
in patients with recurrent fever), but a normal 
elevation within the first 30 minutes occurred in 
the third. Skin temperatures in all three subjects, 
and consequently losses of heat from the body sur- 
face, were elevated, particularly on the extremi- 
ties. Whereas normal subjects can easily tolerate 
50°F air for two hours, the patients with Han- 
sen's disease had considerable difficulty remaining 
at 60°F air for the full two hour exposure. 

When exposed to 100°F air and asked to per- 
form 15 minutes of exercise one of the patients 
failed to sweat to a sufficient extent, rectal tem- 
perature rose and symptoms of insipient circula- 
tory collapse occurred; when heat exposure was 
cautiously attempted a second time several days 
later, similar failure to adjust to heat recurred. 
The two other patients completed a full hour in 
the heat, which included two periods of exercise, 
rather well ; temperature regulation was sufficient 
in these two patients although sweat losses were 
reduced over affected skin areas. 

Radiometric measurements of skin temperature 
were made at 9 bilateral sites (total 18) immedi- 
ately after the patients entered the hot Chamber, 
again 12 to 15 minutes after the first exercise pe- 
riod, and finally 12 to 15 minutes following the 
last exercise period. At sites where affected/non- 
affected areas could be compared, skin tempera- 
ture was higher (up to 1°C) at sites on affected 
areas; this elevation of skin temperature corre- 
sponded to diminution in sweating over the lesion 
areas. 

It is concluded to date that disease involvement 
(Hansen's disease) of approximately one-fourth 
of the body surface area reduces vasomotor adapt- 
ability to moderate cold, with consequent high 
rates of heat loss which are not fully compensated 
for by elevation in heat production so that body 
core temperatures continue to fall. In one patient 
regulation against moderate heat was seriously 
impaired, but heat seemed to be adequately toler- 
ated by the other two patients ; more severe heat 
stress may reveal inadequate regulation in the lat- 
ter patients as well (Drs. Thompson and Buskirk, 
NIAMD, and Dr. Adler, NIAID. 

IV. Studies of Exercise or Work Physiology 

Previously reported studies by this group on 
exercise physiology in normal subjects have been 
extended (by collaboration) to patients with im- 



paired cardiopulmonary function. In this study 
on the action of digitalis, a significant reduction 
has been shown in postexercise oxygen debt by 
digoxin in patients with cardiac disease but with- 
out clinical congestive heart failure. Three pa- 
tients with acquired valvular heart disease and 
cardiac enlargement who were able to perform 
normal everyday activity without difficulty in the 
absence of digitalis therapy were exercised while 
receiving placebo "medication" over an extended 
period and again while receiving digoxin. Di- 
goxin administration did not produce a significant 
change in body weight. Varying degrees of exer- 
cise (7 to 15 minutes walking) were performed on 
a treadmill in the Metabolic Chamber, and oxygen 
consumption was measured continuously before, 
during, and after the period of exercise. The oxy- 
gen debt after exercise which developed during 
digoxin administration was compared to that ob- 
served during placebo administration. 

Inpatients oxygen debt was significantly smaller 
during the period of digoxin administration, al- 
though the treadmill work performed was iden- 
tical. The mean reduction in oxygen debt follow- 
ing 10 minutes walking brought about by digoxin 
was 35%. Work capacity to tolerance with moti- 
vation controlled as closely as possible was also 
increased significantly in all three patients when 
they were on digoxin. Similar studies of oxygen 
debt following exercise were performed with these 
same patients while being given a diuretic (chloro- 
thiazide) alone and again while on digoxin and 
the diuretic together. The effect of the diuretic 
alone was similar to but not as great as that of 
digoxin, and the effect of the two agents together 
was not greater than that of digoxin alone. 

The accumulation of a smaller oxygen debt when 
these subjects were receiving digoxin indicates that 
the functional status of their circulatory system 
was improved by the drug. The results of these 
studies are significant for the long discussed ques- 
tion in clinical management of cardiac patients 
on the value of administration of digitalis in such 
patients in the absence of frank signs of congestive 
failure. From these studies digitalis administra- 
tion would appear to be beneficial to at least some 
patients who have cardiac disease with enlarge- 
ment and who also have some decrease in cardiac 
reserve but without signs symptoms of heart 
failure (Drs. Kahler, NHI, Thompson and Bus- 
kirk, NIAMD, Frye and Braunwakl, NHI). 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 

DISEASES 



INTRAMURAL RESEARCH PROGRAM 

The intramural program in 1962 achieved a bal- 
ance and a maturity that have been sought for the 
last 7 years. With the appointment of Dr. Mau- 
rice Landy as Chief of the Laboratory of Immu- 
nology, succeeding the late Jules Freund, all ten 
laboratories now possess experienced leadership. 
With few exceptions, all section head positions 
within the laboratories are occupied. In addition 
to these more senior places, the Institute intra- 
mural staff consists of younger scientists of accom- 
plishment as well as a group of carefully selected 
junior members. A highly skilled technical staff 
supports the increasingly complex research 
procedures. 

Coincident with the fulfillment of staffing plans, 
the direction of research programs is now well 
established in a course designed to lead to original 
and influential scientific information. NIAID 
clinical investigations have reached a stage of high 
productivity. Projects concern subjects of special 
interest to the Laboratory of Clinical Investiga- 
tions, and also cooperative endeavors with scien- 
tists in other laboratories whose studies have a 
clinical component. This combination of research 
information derived in the laboratory and from 
patient study has resulted in new knowledge of 
precision and usefulness. 

The Middle America Research Unit of the Lab- 
oratory of Tropical Virology located in the Pan- 
ama Canal Zone is now, in its fifth year, a 
well-functioning organization of 38 NTH staff 
members and 8 U.S. Army members. Its labora- 
tory facilities and technical staff are well devel- 
oped and able to handle a variety of viral and 
other microbial agents. The building in which 
MARU is located has undergone recent renova- 
tions which provide additional laboratory space, 
a small clinical area, library area, and a more 
efficient office working area. 



After several years, laboratory renovations in 
Bethesda providing new research facilities are, in 
effect, complete. Each laboratory has well- 
equipped space including special transfer or in- 
strument rooms and animal rooms designed for the 
special needs of each project. The last group to 
accomplish this will be the Laboratory of Im- 
munology, which is now completing the renovation 
necessary to support its activities. Necessary in- 
strumentation is also readily available. 

Deserving of special mention is the new experi- 
mental animal building and insectary now being 
constructed at the Rocky Mountain Laboratory, 
Hamilton, Montana. This structure will provide 
rooms designed to house highly infectious animals 
safely. It also will provide especially designed 
rooms for rearing arthropods and for studying 
transmission of microbial agents by arthropods. 
These modern facilities, of the most advanced 
design, will make it feasible for the Rocky Moun- 
tain Laboratory investigators to conduct many 
studies which have been difficult or impossible 
under previous conditions. Much needed expan- 
sion of the RML library is also under way. 

The Board of Scientific Counselors met in July 
at the Rocky Mountain Laboratory. It had met 
there 3 years earlier and several Counselors at- 
tended both sessions. The Counselors found the 
research program of the Rocky Mountain Labora- 
tory better focused, more mature and with a more 
clearly defined mission than formerly. The Coun- 
selors' advice was helpful, as usual, in clarifying 
overall objectives. 

The Institute has continued its interest in main- 
taining organized research projects, as well as in- 
dividual scientists, in overseas locations where in- 
vestigations can profitably be done. These have 
comprised the Middle America Research Unit in 
the Panama Canal Zone and the field project on 
simian malaria in Kuala Lumpur, Malaya. On 
July 1, 1962, a new Section entitled the Pacific 

193 



194 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Research Section of the Laboratory of Infectious 
Diseases was established in Honolulu, Hawaii. 
Four NIAID staff members are investigating 
eosinophilic meningitis in the Pacific Islands and 
will initiate studies on certain virus diseases 
which appear to be locally important. The Sec- 
tion occupies space at the Queen's Hospital, 
which furnishes, under contract, technical and 
maintenance personnel and research services. At 
the end of 3 years, an evaluation of the need for 
further continuation of this Section will be made. 
These activities, as well as those at Rocky Moun- 
tain Laboratory, and at Atlanta and Columbia, 
South Carolina, provide the Institute with a num- 
ber of satellite laboratories which broaden its 
opportunities for research and ensure close touch 
with problems of importance. 

Intramural scientists, as in previous years, have 
worked in a variety of other laboratories around 
the world. Dr. Jacob Brody of the Laboratory 
of Tropical Virology worked for six months at 
the Institute of Poliomyelitis, Academy of Medi- 
cal Sciences, U.S.S.R., Moscow under the Direc- 
tor, Professor M. P. Chumakov. This was ar- 
ranged under the international agreement for ex- 
change scientists between the U.S.S.R. and this 
country. Dr. Karl Habel, Chief, Laboratory of 
Biology of Viruses accepted an appointment as 
Visiting Professor at the University of Glasgow 
for three months in the spring. He later worked 
at the Karolinska Institutet, Stockholm, Sweden 
with Dr. George Klein on basic virologic prob- 
lems. Dr. Paul Weinstein is conducting collab- 
orative research in filariasis at the National In- 
stitute for Medical Research in Mill Hill, London, 
England. Dr. Sanf ord Stone after spending some 
months with Dr. Halpern, Hopital Broussais, 
Paris, is now working also at the National Insti- 
tute for Medical Research, Mill Hill, London. Dr. 
Louis Olivier is detailed for 2 years to the World 
Health Organization Headquarters in Geneva to 
serve as leader of the WHO bilharziasis advisory 
team. Dr. John Utz is conducting research on 
fungus diseases in the laboratories of Drs. Seg- 
retain and Martin at the Pasteur Institute, Paris. 
Dr. Bill Hoyer of the Rocky Mountain Labora- 
tory is assigned to the Carnegie Institution of 
Washington for special studies on the chemical 
components of viruses and virus surfaces. 



Overseas research programs have been sup- 
ported in part through the use of counterpart 
funds available under Public Law 480. Dr. Ken- 
neth Warren, Laboratory of Parasitic Diseases, 
is currently investigating clinical-laboratory prob- 
lems on schistosomiasis at the Universidade de 
Bahia, Brazil, where he holds a position as Visit- 
ing Professor of Medicine. Other studies under 
the Laboratory of Parasitic Diseases which were 
begun this last year include one on toxoplasmosis 
with the Ministry of Health, Israel and one on 
therapy of schistosomiasis with the High Institute 
of Public Health, Alexandria, Egypt. These ar- 
rangements permit NIAID investigators to con- 
duct studies related to their specific programs in 
favorable locations. In addition, they provide 
opportunities for contact with investigators in 
other countries and an avenue for continuing ex- 
change of scientific information. 

Once again a significant proportion of the In- 
stitute resources has been devoted to offering op- 
portunities for research experience to recent doc- 
toral graduates. Clinical associates appointed in 
the Laboratory of Clinical Investigations have 
participated in laboratory programs not only in 
LCI but to some extent in collaboration with pre- 
ceptors in other laboratories. This collaboration 
was particularly evident with the Laboratory of 
Immunology which has many similar interests and 
occupies space adjacent to the patient care area. 
As in the past, four research associates were 
assigned to other laboratories for full-time labo- 
ratory or fieldwork. Under the new provision for 
appointment of the NIH Staff Fellows, one was 
appointed in the Institute for a limited period of 
time. 

The Institute has appointed its largest number 
of Visiting Scientists this year, a total of seven. 
These scientists represent a wide variety of scien- 
tific disciplines and geographic origin. 

The earlier hope that an Office of Medical 
Biometry would prove useful to the staff has been 
realized. The Institute's biometrician has offered 
valuable consultation and advice to laboratory and 
field investigators, and has originated a number of 
statistical projects. 

Virologists in the Laboratory of Infectious 
Diseases have worked closely with the Institute's 
Collaborative Program in support of the Vaccine 
Development and Viral Reference Reagents activi- 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



195 



ties. The Vaccine Development Program is an 
actual outgrowth and logical goal of much of the 
work which goes on in virologic and field investiga- 
tions. The Reference Reagents Program with the 
aid of Institute virologists and others eventually 
will provide investigators with reference and 
working antisera and antigens to accelerate and 
simplify the normal conduct of virus disease re- 
search. 

Seventy-five years ago research in infectious 
disease within the Public Health Service began 
under the direction of Dr. J. J. Kinyoun at the 
Marine Hospital, Staten Island, New York. This 
event was celebrated at the NIH on November 29, 
1962, with a day and evening meeting attended by 
staff, friends, and former Institute researchers. 
The current activities of this Institute are in many 
ways a direct descendant of that early work. The 
following review reflects the diverse interests of 
NIAID investigators and indicates the maturation 
of a broad research endeavor aimed at carefully 
selected key questions confronting microbiology 
and immunology today. 

ROCKY MOUNTAIN LABORATORY 

An assessment of current and future projects 
was made last spring, first by the scientific staff 
of RML, and then by the Director's Office at RML 
and NIAID. Later the Scientific Counselors 
evaluated the research activities in light of facili- 
ties, opportunities and needs. The deliberations 
resulted in a clarification of the RML mission as 
two interdependent areas of research : ( 1 ) the bi- 
ology of animal and arthropod-borne microbiota 
and (2) immunologic, chemical, and physical phe- 
nomena associated with antigens and microbial 
constituents. 

Rocky Mountain Spoiled Fever 

Case surveillance for Rocky Mountain spotted 
fever has now been intensively resumed through 
various public health agencies. Some explana- 
tion will be sought for the apparent 65% drop in 
reported cases during the decade beginning in 
1949, but it appears unlikely that natural causes 
are the major explanation. It would be useful to 
obtain confirmatory evidence that wide use of 
broad-spectrum antibiotics played a part in the 
sudden reduction in both morbidity and mortality. 

A study of virulence of strains of the etiologic 



agent, Rickettsia rickettsi, revealed that 42 of 43 
isolates from ticks in eastern Montana were M 
strains. These, like U strains, do not produce 
signs of infection in guinea pigs or rhesus mon- 
keys, but unlike U strains, they lack capacity to 
induce the formation of antitoxin against virulent 
strains. Both forms, however, induce high CF 
antibody titers in guinea pigs. The M strains are 
capable of intranuclear parasitism, but can be dif- 
ferentiated biologically from other rickettsias of 
the spotted fever group. Thus, the M isolates are 
considered to constitute a fifth group of R. rick- 
ettsi, additional to the R, S, T, and U strains pre- 
viously characterized. The implications relative 
to mild human infection in the area remain to be 
assessed. 

In addition to the finding, in western Montana, 
of complement-fixing antibodies in 46 to 71% 
of ground squirrels and chipmunks, and in 43% 
of snowshoe hares, 6 virulent strains were isolated 
from spleens of young chipmunks and a golden- 
mantled ground squirrel and, quite unexpectedly, 
from blood of a young snowshoe hare. These are 
the first isolations of R. rickettsi west of the Mis- 
sissippi from naturally infected animals other 
than man. 

Tick Paralysis 

Certain female ticks of the species, Dermacentor 
andersoni hi the Rocky Mountain region are capa- 
ble of producing paralysis in hamsters when fed 
on them. Attempts to inbreed the factors which 
governs this is difficult because males must be 
selected at random. But, genetic determinants are 
possibly involved because this capacity was ac- 
quired by some females in the first to third filial 
generations from inbred males of a paralysis pro- 
ducing stock with females unable to produce 
paralysis. 

Panama Mite Studies 

This project was conducted by two RML staff 
members working in Panama at the Middle 
America Research Unit of the Laboratory of 
Tropical Virology. During the year, 406 pools 
of mites and a few ticks from 639 wild vertebrates, 
mostly rodents, were airmailed to RML for testing 
Ooxiella burneti was recovered from one chigger 
pool from the common spiny rat and inapparent Q 
fever infections occurred in guinea pigs inoculated 



705-685—63- 



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196 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



with two other mite pools, one from bats, the other 
from rodents. Spontaneous infection in test ani- 
mals was ruled out. 

Although Rocky Mountain spotted fever has 
occurred in the Republic of Panama, the first 
strain of Rickettsia rickettsi ever isolated from 
within the Canala Zone was recovered from three 
pools of immature Amblyomma and Haemaphy- 
salis. In addition, two viruses were isolated but 
they had such low virulence that characterization 
has not yet been accomplished. 

A significant augmentation of knowledge of the 
acarine fauna of Central America resulted from 
this project. 

Colorado Tick Fever 

Substantial progress has been made in an under- 
standing of the ecology of this rather widespread 
disaese in the Rocky Mountain area. It was a 
surprise to discover serological evidence of past 
infection in 5 of 49 snowshoe hares in the environs 
of Ottawa, Canada, which is well beyond the 
known endemic area. However, virus could not 
be isolated from 38 pools of 2 species of indigenous 
ticks and further studies are planned to recover 
the agent responsible for antibodies in this area. 
Hares experimentally infected by bites of nymph- 
al Dermacentor andersoni ticks developed signifi- 
cant viremias for 4 days, but only transiently when 
inoculated with large doses of virus. 

Two doses of a refined formalinized vaccine, 
prepared from brains of infected suckling mice, 
produced antibodies in 90% of human volunteers 
without causing ill effects or sensitization to brain- 
tissue antigen. Such a vaccine could be useful in 
areas of high exposure in the field or laboratory. 

Phlyctenulosis 

The study of widespread disability among 
Alaskan Eskimos due to phlyctenulosis with re- 
sultant corneal scarring has been completed. The 
findings are consistent with the hypothesis that 
the disease is primarily a manifestation of hyper- 
sensitivity to infection with Mycobacterium tuber- 
culosis. The scarring is reversible to a greater 
degree than previously suspected, and protection 
is afforded during isoniazid medication, but disap- 
pointingly, not after discontinuance of the drug. 



The Encephalitides 

Results of field investigations on Powassan virus 
and California encephalitis virus indicate that a 
variety of small mammals may serve as natural 
hosts. Serologic evidence of infection with Cali- 
fornia virus was found in snowshoe hares, golden- 
mantled ground squirrels, raccoons, porcupines 
and marmots. Microtus and Peromyscus appeared 
to be the more important hosts of Powassan virus. 
Three more isolates of California virus were made 
from Dermacentor andersoni but its role as a vec- 
tor has not been determined. As yet, the natural 
cycle of these agents remains obscure. 

From our own and others' observations on the 
ecology of western equine encephalitis virus 
(WEE) it appears that the Culex tarsalis-bird 
cycle may be relatively unimportant in the main- 
tenance of this virus. For the third consecutive 
year, virus could not be recovered from G. tarsalis 
taken in the endemic area in North Dakota, and 
this year G. tarsalis from the Vale, Oregon, study 
area were free of virus whereas this species had 
high infection rates the two previous years. Fur- 
thermore, the biology of G. tarsalis in the spring 
and fall is not conducive to the spread or main- 
tenance of virus. 

Q Fever 

The successful separation of cesium chloride 
gradients of Phases I and II in mixtures of Goxi- 
ella burneti has provided, for the first time, a tool 
for the closer control of refined preparations of 
vaccines and diagnostic reagents. This finding 
also provides a possible clue to past discrepancies 
in results formerly thought to have been due to 
strain differences. The radioisotope precipitation 
test developed at RML also has provided a rapid, 
highly sensitive serologic test for measurement of 
antibodies in animal and human serums. This test 
should have broad application in research and in 
epidemiologic investigations. 

Q fever vaccines made from Phase I rickettsias 
have been shown to be 100 to 300 times more potent 
than Phase II in protecting guinea pigs against 
Q fever. As demonstrated by studies in human 
volunteers, a dose of 10 complement-fixing units 
of a refined Phase I vaccine can be given safely 






NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



197 



to persons who are not sensitive to 0. bur-neti. 
Efforts to extract a protective antigen that can be 
used safely in sensitized persons have been unsuc- 
cessful, although information about the chemical 
composition of the organism was obtained. 

Selected Zoonoses of Regional Importance 

The persistence of Pasteurella tularensis in nat- 
ural waters for long periods is still a subject of in- 
terest. One naturally contaminated stream in the 
valley has been positive during all months of the 
year except August and September. Experimen- 
tally, it was determined that the organism survives 
longer at temperatures above freezing and remains 
viable for longer periods in creek water than in 
any of seven other media. At present, scientists 
in this country and those abroad have agreed that 
two types of P. tularensis do exist. All efforts to 
enhance the virulence of the mild type have failed. 

To date, 25 isolates of rabies virus have been 
recovered from bats since studies were initiated in 
1954. In studies on the pathogenesis of rabies in 
mice, virus was found to persist in the brain for 
15 days and antibodies coexisted with virus during 
the late stages of the disease. Most significant 
was the finding that intraperitoneally injected 
street virus will protect against the more invasive 
fixed virus, or against virus injected at the same 
time or later by a more lethal route. 

Viruses and Chronic Disease 

In this new project in comparative pathology, 
the phenomenon of chronic viral infection, as ex- 
emplified by three natural diseases of animals, is 
being investigated to provide some insight into 
pathogenic mechanisms which may be involved in 
vertain chronic diseases of man. To date, the study 
has been limited to exploratory work on scrapie in 
mice, although studies on Aleutian disease of mink 
and chronic pneumonitis of sheep are planned. 
Mice became infected with scrapie virus after inoc- 
ulation by intraperitoneal, intracerebral, subcu- 
taneous, or intranasal routes. Mice, 21 days old, 
inoculated intracerebrally with a 10 -1 dilution of 
scrapie-infected mouse brain suspension, began 
showing clinical signs of scrapie 111 to 135 days 
later. From time of onset, the course of disease till 
death varied from 40 to 50 days. Virus was found 
in the brain in at least the 10~ c dilution, in spleen 
in a 10~ 6 dilution, in thymus in a 10~ 5 dilution, and 
in liver in a 10" 1 dilution. In general, the neuro- 



pathologic changes in mice resemble changes char- 
acteristic of scrapie in sheep and goats. Neither 
inclusion bodies nor micro-organisms were seen in 
in strained impressions of infected brain. Also, 
bacteria, including PPLO, were not cultured from 
infected brain. 

Allergy, Its Mechanisms and Role in Disease 

Studies in allergy were directed toward further 
explanation of the relationship between delayed 
hypersensitivity and the immune process, particu- 
larly the specificity and physiology of mecha- 
nisms involved in certain immunologic phenom- 
ena. While thyroxin is reported to enhance anti- 
body production, this hormone does not affect the 
formation of delayed hypersensitivity and, con- 
trary to previous reports, it did not have a stimu- 
latory effect on antibody production in guinea pigs 
inoculated with diphtheria toxoid. However, 
both antibody production and delayed hypersensi- 
tivity were depressed in pyridoxine-deficient 
guinea pigs. 

In further studies on the specificity of toler- 
ance, delayed hypersensitivity, and antibody pro- 
duction, tolerance was first induced in guinea pigs 
by "gastric feeding" of contact haptens, such as 
dinitrofluorobenzene (DFB) , dissolved in corn oil. 
These tolerant animals did not develop antibody 
or delayed hypersensitivity to the hapten when 
sensitized with the hapten alone. However, when 
subsequently inoculated with a conjugate of DFB 
such as dinitrophenyl-hen egg albumin (DNP- 
HEA), guinea pigs did develop delayed hypersen- 
sitivity to HEA and circulating antibody to the 
hapten. However, they remained unresponsive to 
contact testing with the hapten DFB. When ani- 
mals sensitized with DNP-HEA are subsequently 
"gastric- fed" DFB, they do become tolerant to the 
hapten by contact testing, even though antibody 
to DNP is present. These findings indicate that 
tolerance and delayed hypersensitivity have simi- 
lar specificities directed toward the basic protein 
whereas the specificity of circulating antibody is 
directed toward terminal groupings of the antigen 
(haptens). 

Fine Structure of Microorganisms 

As in the past, studies in this area are made in 
collaboration with numerous guest workers who 
are interested in using RML-developed biophysi- 
cal methods of studying the fine structure of mi- 



198 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



croorganisms varying in size from Protozoa to 
trachoma virus. The major emphasis this year 
was on a study of endotoxin derived from cell 
walls of Bordetella pertussis and the development 
and application of a practical KBr linear gradi- 
ent. By phenol treatment of purified cell walls of 
B. pertussis, an extract was obtained which, in re- 
gard to chemical composition and host reactive 
properties, resembled a typical endotoxin. 

A simple KBr gradient was developed whereby 
microorganisms and fractions thereof could read- 
ily be purified from crude material such as yolk- 
sac suspensions. Organisms contaminated with 
extraneous material are merely dispersed in a KBr 
solution of appropriate density. As the dispersed 
materials seek their own relative densities during 
centrif ligation, sharp bands form in regions where 
the density of each material is equal to that of the 
KBr gradient. This method has been used suc- 
cessfully for separating cell walls from intact 
cells, and for isolating trachoma virus and ricket- 
tsias from yolk-sac suspensions. About 25 times 
as much material can be processed in a single 50-ml 
tube as is possible in other linear gradients. 

Structure and Biologic Activity of Endotoxins 

A more definite characterization of endotoxin is 
necessary before protective antigen can be sepa- 
rated from the endotoxin complex. Our previous 
findings indicated that biologic activity was de- 
pendent upon a macromolecular complex of criti- 
cal size. In further studies on the inactivation of 
endotoxin by human plasma enzymes, the results 
substantiate the previous findings. Thus endo- 
toxin can exist in aggregates ranging from macro- 
scopic particles to those of a minimum size of 10 
Svedberg units. Upon degradation of endotoxin 
with acid or enzymes, only particles the size of 
polysaccharide hapten (1 to 2 Svedberg units) are 
produced. 

On the basis of our own studies on cellulose, 
chitin, and peptides and on others reported in the 
literature, it appeared that endotoxin may consist 
of a micellar system. This arrangement would ex- 
plain the existence of particles having sedimenta- 
tion constants ranging from 190 S or higher (fila- 
ments) to 10 S (micelles, i.e., bundles of chain 
molecules) but no units having S values between 
10 S and 1.4 S (free chain molecules). Accord- 
ingly, the 10 S units, the smallest active particles, 
then would exist as fringed micelles which read- 



ily aggregate to form filaments. These filaments, 
independent of the chemical nature of the chain 
molecules, consistently are 50-100 A. thick. In- 
deed, electron microscopic studies of endotoxin re- 
vealed the existence of fibrils 50-100 A. thick and 
varying lengths. In addition to the fibrils, rodlets 
100-200 A. in length were observed which corre- 
spond to the 10 S units and which were morpho- 
logically identical with the micelles we described 
for regenerated cellulose. If the 10 S particles 
actually were units of the filamentous endotoxin 
and had a fine structure similar to that of the cel- 
lulose micelles, the effect of acid or enzymes on 
endotoxin would be analogous to that produced on 
cellulose micelles after nitration wherein the 
micelles are split into their polysaccharide ele- 
ments, i.e., the chain molecules of which they are 
composed. 

Biologic Properties of Bordetella Pertussis 
Antigens 

Progress has been made in the purification of 
the protective antigen of Bordetella pertussis. 
Heretofore, small quantities of this antigen could 
be isolated by certain electrophoresis and column 
chromatography but much larger quantities can 
now be isolated by starch block electrophoresis. 
As shown by agar gel diffusion tests, the purified 
product contained one main antigen whereas the 
starting material contained 12 antigens. All 
efforts to separate this protective antigen from 
the histamine sensitizing factor have failed. Both 
are found in identical electrophoretic fractions 
and they are precipitated by similar concentra- 
tions of acetone and at the same pH ; both activ- 
ities are located in the cell wall and the protective 
activity of vaccines correlates with their ability 
to sensitize mice to histamine. For these reasons, 
both activities are believed to be associated with 
the same substance in the cell walls. 

Biologic Behavior of Microbial Proteins and 
Nucleic Acids 

Results of further studies on radioisotope pre- 
cipitation (RIP) tests indicate that this is an 
extremely useful tool for investigations concern- 
ing surface characteristics of viruses and their 
protein constituents. Because of the extreme sen- 
sivity and specificity of the test, minor alterations 
in poliovirus protein induced by heat, ultraviolet 
light, or lyophilization could be detected. With 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



199 



this test, it was also determined that antibody 
directed toward whole tobacco mosaic virus 
would not readily combine with virus protein ob- 
tained by acetic acid dissociation. 

The sensitivity of the RIP test was compared 
with that of the tissue culture neutralization 
(TCN) test and the plaque-forming-unit-precipi- 
tated (PFTJP) test. In these comparisons, known 
numbers of antibody molecules and virus particles 
were used and the ratio of the first to the second 
at antibody endpoint could be determined. In 
the TCN test this ratio was 100,000 to 1, in the 
RIP test it was 5 to 1, and in the PFUP test it 
was 700 to 1. 

Although type II poliovirus was originally used 
for development and standardization of the test, 
equally reliable results have been obtained with 
types I and III. The test is now being used in 
studies concerning the serology of measles, psit- 
tacosis, and trachoma. 



LABORATORY OF GERM-FREE 
RESEARCH 



ANIMAL 



The studies in this laboratory have the general 
objectives of establishing the role of the flora (or 
of a particular organism) in some broader disease 
problem or biological process, and evaluating the 
latter in the absence of microbial influence. Sev- 
eral aspects of these studies are at or nearing com- 
pletion. 

Penicillin Not Toxic for Germ-Free Guinea Pigs 

One interesting finding during the year empha- 
sizes the unique value of the germ-free animal as 
an experimental tool. Among the poorly under- 
stood reactions to penicillin is the extreme sensi- 
tivity of guinea pigs to this antibiotic. They may 
succumb following the injection of dosages which 
are small fractions or those required to kill other 
animal species and are at levels not generally con- 
sidered to be toxic. One explanation for this 
anomalous circumstance has been that the anti- 
biotic may upset some microbial balance in the ani- 
mal (since such small dosages can hardly eliminate 
all the organisms) and a particularly virulent 
species may overgrow others and kill the animal. 
However, there has been little evidence to support 
this or other possibilities. We have found, in col- 
laboration with LCI, that germ-free guinea pigs 
show no apparent ill effects following injections of 



32,000-64,000 units of penicillin per 300-gram ani- 
mal. Not a single death has occurred among sev- 
eral litters inoculated. However, such dosages 
generally result in the death of some or often the 
majority of conventional guinea pigs a few days 
after injection. These findings point strongly to 
the likelihood that the untoward effect of the anti- 
biotic is exerted indirectly through some effect on 
the flora rather than an expression of a direct toxic 
action. 

Death of Germ-Free Guinea Pigs 

In other studies with germ-free guinea pigs it 
has been observed that these animals will invaria- 
bly die when removed from the isolator and placed 
in an unsterile environment such as an animal 
room. This is not true of other germ-free animals. 
Death occurs within 2 days and, no particular 
contaminating organism has been incriminated 
consistently, as yet. In efforts to ascertain factors 
involved in this unusually high susceptibility, var- 
ious protective measures Avere attempted including 
previous mono-infections, antibiotics, sulfa drugs, 
etc. Most have been unsuccessful in reducing 
mortality after removal from the isolator. How- 
ever, some prolongation of survival time and 
occasional survival have recently been obtained 
with prior treatment with staphylococcal endo- 
toxin. Further results in this direction can pro- 
vide some insight into the role this substance plays 
in stimulating "nonspecific" resistance. 

Other studies on the immune response in guinea 
pigs without a flora have indicated that they do 
not produce antibody to an antigen such as BGG 
as early, or as in high titers, as do their conven- 
tional counterparts. 

Pulmonary Tumors 

The first part of the study of the comparative 
incidence of methylcholanthrene-induced pulmo- 
nary tumors in germ-free and conventional mice, 
(i.e., those living in a contaminated environment) 
were concluded. The results indicated the same 
incidence in germ-free mice, their genetic controls 
maintained under usual animal room conditions, 
and NIH General Purpose mice. The mean nod- 
ule count was at least as high in the germ-free ani- 
mals and in some instances higher than in the 
General Purpose mice. In addition to these 
tumors, subcutaneous sarcomas and lymphocytic 
leukemia were also produced in the germ-free mice 



200 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



at about the same rate as in the General Purpose. 
Our information on the viral status of the germ- 
free mice is incomplete, in view of the large num- 
ber of viruses reported to occur in mice. Serologic 
and other tests conducted in collaboration with 
LID for polyoma, K virus, mouse adenovirus, 
Theiler's GD VII, PVM, Keovirus and MHV 
have been negative with one possible exception, 
Keovirus type 3. It has not been possible to re- 
cover this virus from the germ-free colony. Mice 
from the General Purpose colony have shown a 
high incidence of antibodies to at least three of 
these viruses. Of course, little can be said of the 
role, if any, that viruses may have played in these 
experiments. However, it is of interest that pul- 
monary tumors were induced in these germ-free 
mice at least as well as in mice exposed to a varied 
microbial flora including several viruses. A dif- 
ference, bordering on significance, was noted in the 
incidence of one of the other tumors among the 
groups. However, the overall incidence was so low 
that further studies are indicated. 

Death in Conventional and Germ-Free Mice 

A continuing study of the longevity and post- 
mortem findings in germ- free and conventional 
mice of the same genetic stock maintained on the 
same diet has provided data on over 100 animals. 
It appears, thus far, that the causes of death, 
where established, are essentially similar in both 
groups, with tumors apparently accounting for the 
majority of deaths. Most of these were pulmo- 
nary neoplasms ranging from benign adenomas to 
widely disseminated carcinomas. A nephropathy 
observed in some of the conventional animals, and 
considered by some to be associated with response 
to infection, was also noted in similar incidence 
in germ-free animals. It should be pointed out 
that the conventional controls for our germ-free 
mice are probably similar to the so-called specific- 
pathogen-free animal. They are not exposed to 
as many pathogenic bacterial or parasitic infec- 
tions as other colonies might encounter. However, 
they do harbor most of the usual bacterial con- 
taminants found in mice. 

Tissue Reactions to Mouse Nematode 

Histopathologic comparisons of infections with 
the mouse nematode, Nematospiroides dubius, in 
germ-free and conventional mice appear to have 
resolved a question that has been raised by several 



investigators. Parasite larvae penetrating host 
tissues from a contaminated site, either from the 
intestinal lumen (as in the case of N. dubius) or 
from the external environment, can carry bacteria 
and other organisms along with them. It has been 
the feeling of some that the host's tissue reaction 
around the invading larvae is, in these instances, 
primarily a response to the bacterial contaminants. 
Some have even fed the host antibiotics to demon- 
strate this point. A study of the lesions and tissue 
reactions around N. dubius in the intestinal wall, 
from the initial penetration of the larvae through- 
out their development, has shown no significant 
difference between the germ-free host-fed sterile 
larvae and a bacterially-contaminated host-fed 
contaminated larvae. 

Thyroiditis 

Autoimmune thyroiditis has been found to per- 
sist at least 18 months, and in some cases as long 
as 2 years after a single immunizing dose of thy- 
roid adjuvant. This is in contradistinction to 
reports of others, who have found that the disease 
disappeared a few months after its production in 
guinea pigs, and may be a function of the immu- 
nizing procedure. 

At 6 months and later periods, some decrease in 
the delayed hypersensitivity to thyroid antigen, 
as determined by skin test, has been found, with a 
modified type of reaction best described at present 
as an "intermediate" type. This appears later 
than the immediate Arthus reaction and disap- 
pears earlier than the classical delayed tuberculin 
reaction. This new type of skin reaction is being 
investigated further. 

LABORATORY OF BIOLOGY OF VIRUSES 

The scientific work of the laboratory during the 
past year has been most productive and can read- 
ily be divided into five areas of basic research 
related to viruses: immunology, genetics, cell 
metabolism, biochemistry of viral replication, and 
biophysical studies. 

Immunologic Studies 

Investigations aimed at evaluation of factors 
responsible for recovery of animals and man from 
primary, acute virus infections have raised ques- 
tions concerning the role and importance of spe- 
cific antibodies and hypersensitivity in this 
situation. Other non-specific factors including 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



201 



elevated body temperature and interferon have 
been shown to be more effective than antibodies in 
recovery of guinea pigs from local vaccinia virus 
infection and the fatal encephalitis produced in 
mice by encephalomyocarditis virus. 

Studies of the synthesis of viral proteins in the 
poliovirus-HeLa cell system have led to the dis- 
covery of a new antigen in polio-infected cells 
which is characteristic of dissociated viral protein 
subunits, but is not a part of the mature virus par- 
ticle. This new antigen appears early in the virus 
multiplication cycle. This same project is accu- 
mulating evidence on the physicochemical char- 
acterization of the subunit of the virus coat which 
determines the immunological specificity of the 
virus and therefore is directly involved in the 
antigenicity of poliovirus vaccines. 

That cells transformed to tumor cells by poly- 
oma virus contain a new foreign cellular antigen 
and immunological reaction to that antigen can 
limit the oncogenic potential of this virus was first 
demonstrated in this laboratory. It has now been 
shown that this phenomenon is also true in the 
SV40 and Moloney leukemia tumor virus systems. 
Furthermore, other laboratories have found it to 
be true with Rous sarcoma, Gross leukemia and 
rabbit papilloma so there is good reason to expect 
that it may be characteristic of all virus-induced 
tumors. One important recent contribution is the 
demonstration that these new cell antigens appear 
specific for the tumors produced by each virus. 
This means that a method is now available to 
prove etiological relationship between a given 
virus and a given tumor, provided the tumor is 
transplantable in adult animals. 

Genetics 

A continuation of studies on sulfated poly- 
saccharide virus inhibitors has shown that growth 
of certain virus mutants is not only uninhibited 
by these materials, but is actually enhanced. Thus 
the current live attenuated poliovirus vaccine 
strains were found to contain three plaque types 
of particles and the use of dextran sulfate makes 
their demonstration easy. The possibility that 
these mutants may vary in their neurovirulence 
suggests interesting practical implications in the 
vaccine field. 

The use of viral susceptibility as a genetic mark- 
er for cells in tissue culture has led to some pioneer- 



ing results in the newly developing field of human 
cellular genetics. Mutant cells from cultured 
human amnion have been found completely resist- 
ant to infection with polio- virus, although derived 
from completely susceptible parent cells. The 
basis of this resistance is lack of adsorption of 
virus to the cell, and the mutation rate for this 
character has been determined. 

Cell Metabolism 

Work has continued on the definition of the 
enzymatic basis for resistance of mutant HeLa 
cells to the toxic action of 2-desoxyglucose. It has 
been shown that certain steroids will increase the 
tolerance of susceptible cells to this toxic glucose 
analogue. 

A possible new type of RNA has been found in 
mammalian cells in tissue culture. This is an acid- 
soluble, nondialyzable RNA which is labeled much 
more rapidly by radioactive precursors than are 
the other known types of RNA. Further investi- 
gations are in progress to determine the function 
of this interesting cell material. In parallel 
studies there has been evidence that the effect of 
interferon may be that of inhibiting the synthesis 
of viral RNA." 

Biochemistry of Viral Replication 

The work in this area is in the forefront of the 
field. Although considered here in respect to polio- 
virus and vaccinia virus replication the real sig- 
nificance of the results is much broader than just 
its relation to a particular virus. New informa- 
tion obtained this year on the replication of the 
RNA polio-virus in human cells has shown that- 
normal cellular DNA-dependent RNA synthesis in 
the nucleus stops on infection but cytoplasmic 
RNA synthesis continues. 

A uracil analogue which inhibits virus forma- 
tion when applied early after infection has been 
found not to inhibit synthesis of viral RNA as 
such, but appears to affect production of an earlier 
type of RNA which is only associated with virus 
infection. 

Work with the DNA vaccinia virus indicated 
that cellular and viral DNA are probably synthe- 
sized by different enzymatic systems. Viral DNA 
replication is not necessary for formation of viral 
proteins and the rate of viral protein synthesis is 
independent of the multiplicity of infection. 



202 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Biophysical Studies 

Work in this area during the past year has been 
aimed chiefly at the development of new physical 
methods to study biochemical and biological events 
at the molecular level with greatly increased 
resolution. 

Refinements in the electron microscopic technics 
used for examination of ultrathin sections of cells 
have been developed and are now being used for 
seeking the physical nature of virus precursor 
materials. 

Preliminary results have been very encouraging 
on the development of a technic whereby uranium 
can be combined with nucleic acid, caused to fis- 
sion by exposure in a nuclear reactor and fission 
tracks demonstrated at the molecular level by elec- 
tron microscopic technics. 

LABORATORY OF TROPICAL VIROLOGY 

This laboratory comprises a satellite laboratory, 
the Middle America Research Unit (MARU), lo- 
cated in the Panama Canal Zone and a base facil- 
ity in Bethesda. Investigations are integrated 
into an attack on problems of virus diseases within 
North, Middle and South America. This includes 
ecologic, clinical, diagnostic, and serologic aspects 
of selected disease problems. The Walter Reed 
Army Institute of Research jointly supports 
MARU and has established investigations in my- 
cotic and bacterial diseases. 

Vesicular Stomatitis Virus 

Repeated isolation of vesicular stomatitis virus, 
Indiana type, from Panamanian phlebotomus 
sandflies and the existence of nearby endemic areas 
of human infection, supported by findings of other 
laboratories, establish it as a new arbovirus of 
medical and not only veterinary importance. Hu- 
man disease in one of its forms was illustrated by 
a MARU staff member hospitalized with a febrile 
illness (laboratory infection). 

Sandfly Fever in the New World 

Although the phlebotomus sandfly virus isolates 
are not related to either Sicilian or Naples types 
of sandfly fever, a virus (JW-10), isolated from 
the blood of a Nicaraguan patient with febrile ill- 
ness in Panama, is a member of the New World 
Naples-like complex of viruses. Besides the Na- 
ples virus prototype (Sabin) and our agent, the 



complex includes a virus active in the Belem, 
Brazil, area. With the demonstration of common 
antibodies against JW-10, these findings assume 
potential public health importance. A new phle- 
botomus virus repeatedly recovered during the 3 
years of the Bocas del Toro project (four strains 
from MARU and four from GML), was found to 
be related to a Brazilian (Belem) animal virus iso- 
late, but not to Naples or Sicilian sandfly fever 
viruses. Its public health significance is as yet 
unknown. 

Hemorrhagic Fever in Bolivia 

Field studies of an acute febrile illness with 
hemorrhagic manifestations indicate that over 500 
endemic cases with high mortality have occurred 
in an isolated area of Northeast Bolivia during the 
past 3 years. The clinical picture resembles Ar- 
gentinian hemorrhagic fever. Extensive labora- 
tory studies to clarify the suspected viral nature 
of the etiologic agent are in progress. 

Encephalitis Viruses in Panama 

Pioneer studies on the occurrence of EEE and 
VEE in Panama are beginning to clarify the local 
epidemiology and epizootiology of these virus in- 
fections. While studying an outbreak of EEE 
among horses, the virus of VEE was isolated from 
the blood of 5 to 10 febrile people residing in the 
epizootic area. The December 1962 outbreaks of 
encephalitis, both human and equine, in nearby 
Caribbean countries (Colombia, Venezuela, Cuba 
and Jamaica) add particular urgency and impor- 
tance to the field and laboratory studies. 

Bunyamwera Group of Viruses 

Specific neutralization of Cache Valley virus by 
normal calf serum was demonstrated in cell cul- 
tures maintained with media containing serum 
from Illinois, Indiana, Pennsylvania, New York, 
New Jersey and Maryland calves. Significance 
of the finding Avas corroborated by recent isola- 
tions of Cache Valley virus from U.S. and Pana- 
manian mosquitoes. Apparently this member of 
the Bunyamwera group has been active over a 
large portion of the Western hemisphere. The 
eight prototype members of this group produced 
CPE in tubes and plaques under agar ; hopefully 
this will lead to plaque reduction test as a practi- 
cal serologic tool to investigate the role of these 
viruses in human and animal disease. 



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203 



Pathogenic Agents From Panamanian Aearina 

Although no viruses were isolated from mites in 
a 2-year collaborative project with RML, two 
virus strains of low titer (still unidentified) were 
isolated from ticks ; rickettsiae were isolated from 
both ticks and intradermal chiggers taken off 
Panamian animals. Of major significance was 
the finding of 80 species of chiggers, of which only 
17 were known to exist in Panama and many were 
new to science. 

VEE Vaccination 

Attenuated VEE virus produced by the Biologi- 
cal Laboratories at Fort Detrick for experimental 
human immunization was evaluated at MARU 
under field conditions. The frozen virus (rather 
than the lyophilized material of lower potency) 
produced reasonable serologic responses, presum- 
ably indicative of some immunologic protection, 
and only mild side reactions occurred. Its use for 
the protection of potentially exposed personnel 
will be continued. 

Serologic Reagents From Ascitic Fluid 

Hyperimmune ascitic fluid production in mice 
by virus-adjuvant inoculation has yielded an ex- 
cellent serologic reagent at MARU and ABVS 
with a variety of arboviruses; the procedure is 
being adopted as a basis for commercial contract 
production of the standard antibody reagent for 
national distribution (under the auspices of the 
NIAID Panel for Arboviruses). Polyvalent 
grouping reagents constitute a major need of arbo- 
virologists throughout the world. Polyvalent 
hyperimmune ascitic fluids produced by repeated 
inoculation of mice with antigens and also com- 
bined antiserum pools, made by mixing two or 
more specific antisera, have been successfully made 
and used with several arbovirus groups. This 
may serve as a model for standard reagent produc- 
tion. 

Leptospirosis in Panama 

For the first time in Panama, leptospirae have 
been isolated from humans, small mammals and 
from the contaminated river water which served 
to infect exposed troops. New serological groups 
and types are being designated for these organ- 
isms. 



Bats and Histoplasmosis 

Recovery of histoplasma capsulatum from livers 
and spleens of bats belonging to five genera and 
collected from widely separated harborages, re- 
emphasized the importance of the current study 
on the role of the bat in the ecology of histoplas- 
mosis. Histoplasmin skin test continues to be a 
major epidemiologic tool: conversion to positive 
of 3/77 men in Panama for only 67 days (as part 
of Operation Swampfox II) was thus demon- 
strated ; the first survey of the relatively isolated 
San Bias Archipelago Indians disclosed overall 
positivity of 50% among 1,038 persons tested 
(most reactors between 11 and 20 years of age) . 

LABORATORY OF IMMUNOLOGY 

It is evident in the individual reports that the 
original programs developed in the Laboratory 
of Immunology, as they have widened in scope, 
have generated intensive collaboration among the 
sections of this Laboratory, with other labora- 
tories of this Institute, and with units of other 
Institutes of the NIH. When collaboration with 
investigators outside the NIH offers special ad- 
vantages for prosecution of the work, these, too, 
have been fostered and extended. 

Cross Reactions in Human Malaria Disclosed by 
Fluorescent Antibody 

With the discovery that by immunofluorescent 
methods antibody can be detected and titrated in 
the sera of malaria patients, it has been possible 
to study cross reactions in this disease. Sera from 
volunteers infected with the B strain of Plasmo- 
dium cynomolgi were allowed to react with the ho- 
mologous parasite, and the heterologous parasites 
of two strains of Plasmodium- vivax. Although 
there was considerable cross reaction between the 
two species the maximum antibody titers with the 
homologous parasite tended to be higher than with 
the heterologous parasites of either strain of P. 
vivax. This suggested the possibility that perhaps 
not only certain antigens are shared by the two 
species but that P. cynomolgi parasites may possess 
a different antigen or antigens. The cross reac- 
tions between various species of the malaria Plas- 
modia may contribute to a better understanding of 
the mechanisms involved in immunity to malaria. 



204 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Allergy Reagins Identified as Beta 2 A-Globulins 

The reagins or skin sensitizing antibodies found 
in individuals with atopic allergies are believed to 
be responsible for the specific allergic reactions 
such as hay fever, asthma or various skin reactions. 
The specific removal of the beta 2 A-globulin frac- 
tion of sera from ragweed sensitive individuals by 
absorption with a unispecific anti-beta 2 A-globu- 
lin serum resulted in the loss of all detectable skin 
sensitizing activity. The beta 2 A-globulins have 
been thought to possess antibody activity but there 
has been little direct evidence to support this hy- 
pothesis. The data indicate that the skin sensitiz- 
ing antibodies in these sera were beta 2 A-globulins. 
This approach is promising for the investigation 
of other antibody activities which may be associ- 
ated with the beta 2 A-globulins. 

Physiological Basis for Susceptibility to Ana- 
phylactic Shock 

The pronounced difference in susceptibility of 
Strain 2 and Hartley guinea pigs to acute anaphy- 
lactic shock was not related to their reactivity to 
histamine, but did correspond to differences in the 
histamine content of their lungs. It was found 
that both strains had the same content of fixed 
(lung) histamine; the genetic difference was ex- 
pressed in the amount of histamine which was lib- 
erated in the course of anaphylactic reaction. In 
the susceptible Hartley strain, this was approxi- 
mately seven times as much as in the refractory 
Strain 2 guinea pigs. It may be especially signif- 
icant that the ratio of liberated histamine corre- 
sponded to differences in the number of mast cells 
present in the lung tissue of these two strains. 

Relationship of Delayed-Type Hypersensitivity 
to Allergic Thyroiditis 

The various facets of the immune response to 
experimental allergic thyroiditis in the guinea pig 
have been studied to ascertain which, if any, are 
directly related to this disease. No correlation was 
evident between levels of circulating antibody or 
immediate skin reactivity and the progression of 
the disease. Delayed skin reactivity, on the other 
hand, was found consistently to closely parallel the 
disease during its developmental and declining 



phases; it may, therefore, be a causal factor hi 
allergic thyroiditis. This approach to the study 
of the pathogenesis of allergic thyroiditis may help 
clarify the nature of the disease in man. 

Implications of Allotypy for Experimental 
Biology 

Antigenic differences in sei*um protein allotypes 
among individuals of the same species have been 
investigated further by chemical, immunological 
and genetic methods. The basic work on allotypes 
which originated with studies in the rabbit, have 
now been extended to mice and to primates, in- 
cluding man. The studies in mice are of unusual 
interest since in this species the allotypes serve 
directly as markers for the study of protein 
genetics, tumor immunity, and transplantation im- 
munity. The findings in primates are significant 
inasmuch as they have direct implications for man 
especially with respect to transfusion reactions and 
to maternal-fetal interactions from allotype in- 
compatibility in a manner analagous to the Eh 
situation. 

Maternal-Fetal Interactions Lead to Long-Lasting 
Inhibition of Protein Synthesis in Off- 
spring 

In female rabbits immunized to the allotype of 
the father, the antiallotype antibody in the mater- 
nal circulation specifically inhibited the geneti- 
cally determined synthesis of the paternally de- 
rived allotype of the young rabbits for at least one 
year and was associated with a compensatory in- 
crease in the allelic allotype derived geneticallj 
from the mother. This phenomenon of "allotype 
suppression and associated compensation" is novel 
and may prove to be of fundamental significance 
comparable to the induction of "immunological 
tolerance" of the newborn. In the former situa- 
tion (allotypy) synthesis of a normal protein is 
suppressed, whereas in the latter situation there 
is interference with the synthesis of a new protein 
antibody. The basic mechanism may nevertheless 
be similar in both situations. The phenomenon 
of "allotype suppression," involving as it does 
genetically defined parameters, offers special ad- 
vantages for delving deeper into understanding 
these mechanisms at the molecular level. 






NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



205 



Cellular Production of Gamma Globulin Allo- 
types Shown by Fluorescent Antibody 

The histological sites and the cellular produc- 
tion of the allotypes have been investigated. Rab- 
bit antisera to allotypes A4 and A5 controlled by 
allelic genes were fractionated on a DEAE cellu- 
lose column to isolate the 7S gamma globulin for 
coupling with fluorescein isothiocyanate and liss- 
amine rhodamine B (RB 200) so as to produce 
specific conjugates of contrasting colors. Fluo- 
rescent antibody studies of lymph nodes, with 
mapping of adjacent sections by conjugates to 
either of the two allotypes, and with double stain- 
ing of the same section with the conjugates of the 
same or contrasting color to either of the two allo- 
types, yielded information on how the allotypes 
are produced in the heterozygote adult. No evi- 
dence was found of separate groups or clones of 
lymphoid cells producing only one of the two allo- 
types. Rather, the evidence indicates that the two 
allelic allotypes are produced by the same cell. In 
rabbits with one allotype suppressed by passively- 
transferred antibody, the lymph nodes did not ex- 
hibit the presence of the suppressed allotype in- 
dicating that suppression involves inhibition of 
allotype synthesis. Production of both allelic 
allotypes by one cell indicates that the mechanism 
of suppression cannot be explained as clonal de- 
struction by antibody. Rather this occurs by a 
more subtle action of antibody on the cell which 
forces a shift of synthesis from the paternally de- 
rived allotype to that of the maternally derived 
allotype. 

Allotypes Shown To Be Genetic Markers in Cell 
Transfer Studies 

An issue of special importance in basic work on 
the transfer of homologous lymph node and splenic 
cells is the cellular source of the antibody which 
subsequently appears in the recipient animal. 
Rabbits homozygous for each of an allelic pair of 
allotypes of gamma globulin (A4 and A5) were 
used as donors and as recipients of transferred 
Shigella antigen-incubated lymph node cells. 
Following the appearance of agglutinins to 
Shigella in the sera of recipient animals, the allo- 
type of the agglutinin was determined by absorb- 
ing it to Shigella, treating these agglutinates 
with fluorescein-conjugated rabbit anti-A5-gam- 
ma-globulin and the anti-A4-gamma-globulin. 



The reactions of the recipients' sera were consist- 
ently positive for the donors' gamma globulin allo- 
type but not for their own. With decline of the 
agglutinin level in the recipients, these animals 
were actively immunized with Shigella; the anti- 
body produced then reacted with the fluorescent 
antibody against their own allotype. The find- 
ings show that in such transfer experiments, 
the antibody which initially appears in the re- 
cipient serum is synthesized by donor cells, thus 
illustrating how allotypes can serve as genetic 
markers. In a further use of the allotype as a 
marker, prenatally derived thymus and spleen 
cells have been transferred to prenatal recipients. 
The appearance in the recipient's serum of the 
donor gamma globulin allotype indicated success- 
ful transfer of lymphoid cells to a tolerant host. 
With this experimental method it becomes feasible 
to study the role of the thymus in early life, and 
the onset of immunological competence. 

Genetic Control of Allotypes Related to Chemi- 
cal Structure 

Chemical studies have shown that allelic allo- 
typic specificities do not appear on the same mole- 
cule. On the other hand, non-allelic specificities 
may appear on the same molecule or separately. 
Genes in coupling or repulsion in double heterozy- 
gotes may contribute allotypic specificities to the 
same molecule. Thus far results are analagous to 
the well-known chemical and genetic studies of 
hemoglobin. The chemical studies of the gamma 
globulin allotypes are likewise directed toward 
relating the structural difference in the protein to 
that of the genetic control. Since the gamma glob- 
ulin molecule is complex, sub-units have been 
studied with the objective of obtaining the smallest 
fragment exhibiting the genetic marker. It was 
established that such a small molecular weight sub- 
unit is obtainable, which would be suitable for 
fingerprinting studies to elucidate amino acid dif- 
ferences attributable to allelic genes. 

New Mouse Allotypes Discovered by Ascitic Fluid 
Technique 

Mice of several inbred strains were immunized 
with mouse anti-Salmonella agglutinates. The 
mice developed ascitic fluid containing precipitat- 
ing antibodies to three allotypes. Two of these 
are gamma globulins under the control of allelic 
genes. Coisogenic lines of mice are being estab- 



206 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 






lished which will be useful for studies in tumor 
and transplantation immunity. The availability 
of many known genes in the mouse and the numer- 
ous inbred lines make it feasible to establish the 
genetic linkage group for each allotype locus as 
discovered. 

Allotypes Found in Man 

The first allotypes characterized in man resulted 
from the testing of sera from multitransf used pa- 
tients. These allotypes were found to be the low 
density beta lipoproteins. The experimental ap- 
proach toward finding allotypes in man has been 
via the use of the monkey and the chimpanzee as 
surrogates. This has led to the production of pri- 
mate antisera which have revealed new gamma 
globulins and individual differences among these 
gamma globulins in the sera of normal individuals. 

New Methodology for Characterization of Serum 
and Tissue Enzymes 

Methods have been developed for characteriza- 
tion of amidase and esterase activities of trypsin- 
like, chymotrypsin-like and carboxy trypsin -like 
enzymes in serum as well as in tissue extracts. 
These methods allow the enzyme studies to be 
made after agar gel electrophoresis and even after 
Immunoelectrophoresis, because the enzyme-anti- 
enzyme precipitin bands continue to exhibit en- 
zyme activity. It has thus become feasible to iden- 
tify the electrophoretic mobility, immunochemical 
specificity, and enzymatic activity of serum and 
tissue enzymes in a single laboratory procedure. 
Initial clinical studies with previously developed 
techniques of this kind have shown significant de- 
creases in beta-naphthyl esterase activity in sera 
from patients with Wilson's disease and several 
other liver disease states. These novel methods 
afford new opportunities and applications in the 
increasingly important area of characterization of 
tissue antigens in autoimmune diseases and in tis- 
sue transplantation studies. 

Action of Immunosuppressive Drugs on Normal 
Serum Globulins 

The suppressive effect of antimetabolites on im- 
mune responses has been the subject of intensive 
study in recent years, but there have been no in- 
dications of the effect on antibody proteins in non- 
immunized subjects. In collaborative studies be- 



tween LI and Laboratory of Clinical Investiga- 
tions, the administration of the purine antimetabo- 
lite 6-thioguanine was found to have therapeutic 
effects in patients with the nephrotic syndrome, 
and to produce decreases in serum gamma globulin 
levels in patients receiving this antimetabolite. In 
rabbits both 6-thioguanine and 6-mercaptopurine 
were found to produce decreases in the level of 
serum gamma globulins, but other serum proteins 
were increased. The hypogammaglobulinemia 
produced in both humans and rabbits indicates 
that the effect of the antimetabolites on antibody 
producing cells is not limited to suppression of 
primary response to antigenic stimuli, but is a 
continuing effect on the mechanisms for maintain- 
ing "normal" gamma globulin levels. This work 
indicates that the purine antimetabolites offer 
promising alternatives to the use of corticoste- 
roids in the therapy of nephrosis, and suggest that 
other disease entities associated with autoimmune 
phenomena may also be amenable to such treat- 
ment ; moreover, they suggest approaches to deter- 
mine possible mechanisms by which the antimeta- 
bolites exert their effects. 

Study of Delayed Allergy With in Vitro 
Techniques 

The lack of an in vitro test system to substitute 
for skin testing and other similarly unsatisfactory 
procedures in the intact animal or human has pre- 
vented the evaluation of the role of the delayed 
type of hypersensitivity in the production of tis- 
sue lesions in the autoimmune diseases and in the 
rejection of tissue homografts. Reassessment of 
the "Rich and Lewis" test system for study of 
spleen and lymph node fragments in tissue culture 
is providing encouragement for approaching these 
issues. Migration of cells from explants of tissue 
from sensitized animals is measured in the pres- 
ence of antigen, and specific inhibition of cell mi- 
gration appears to reflect cellular or delayed type 
hypersensitivity. A heretofore unreported serum 
factor appears to confer on normal cells the prop- 
erty of exhibiting hypersensitivity to antigens. 
With this in vitro technique it may be possible 
to ascertain the relationship between circulating 
antibody and delayed-type hypersensitivity, and 
eventually to assess the role of delayed hypersen- 
sitivity in many host phenomena of immunologi 
cal importance. 



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207 



LABORATORY OF CLINICAL 
INVESTIGATIONS 

This is the third year of operation of the Labor- 
atory under the present Clinical Director. During 
this time a number of new programs have been 
initiated, notably that of clinical immunology, res- 
piratory viral disease in volunteers, malaria, 
leprosy, and mechanisms of host response. In ad- 
dition, strong programs continue in systemic fun- 
gus disease, biochemistry, immunology, and other 
activities previously in operation. 

Purine Antimetabolites in Non-Neoplastic 
Diseases 

In recent months a significant new trend in 
medical therapy has been extended by the obser- 
vation that cases of nephrosis, of the type classi- 
fied as of unknown etiology, have been greatly 
benefited by treatment with one of the purine anti- 
metabolites, 6-thioguanine (6-TG). In addition 
to nephrosis, patients with chronic hepatitis and 
systemic lupus erythematosus have now been 
treated with some favorable results. 

At present two patients who had received long 
courses of steroid therapy with varying degrees 
of response and many of the toxic consequences 
of steroid treatment have shown complete remis- 
sion from nephrosis on 6-TG treatment alone or 
with 6-TG plus a previously inadequate dose of 
steroid. In one case, an adult male, whose condi- 
tion was considered hopeless, restoration to ap- 
parent good health and return to full employment 
have occurred. He has been well for more than 
1 year since treatment. The other patient, a 14- 
year-old girl, whose appearance was greatly 
marred by the plethora, striae, and rotundity 
characteristic of high-dose steroid treatment, has 
returned to a normal appearance in association 
with complete remission of nephrosis following a 
short course of 6-TG. She relapsed and was given 
longer treatment with a second remission. 

Several patients with hepatitis were given 6-TG 
but presumably because of their disease, they are 
very sensitive to its toxic effects. Nevertheless, 
one adolescent male has apparently undergone re- 
mission after 6-TG treatment. One patient with 
systemic lupus improved with 6-TG, relapsed and 
then improved on retreatment. This sequence was 
repeated three times. 



Toxicity of 6-Thioguanine 

There is a considerable variation in threshold of 
tolerance of 6-TG, and toxic effects consist prin- 
cipally of agranulocytosis and thrombocytopenia, 
at which time severe infection can be a further 
complication. One severe episode of toxicity has 
occurred complicated by gram-negative bactere- 
mia. The patient recovered. 

A study in rabbits was undertaken of the effect 
of 6-TG on protein synthesis and antibody forma- 
tion. It revealed a significant reduction in serum 
gamma globulin following 6-TG. The same find- 
ings as well as a significant reduction in isohemag- 
glutinin titers were observed in nephrotic patients 
given 6-TG. 

Action of 6-Thioguanine 

Many observers believe that some cases of 
nephrosis, hepatitis, lupus, some anemias, and a 
number of other diseases are due to the effect of 
antibody produced in the host to its own body 
constituents such as liver, kidney, platelets, con- 
nective tissue, etc. The initiation of such a reac- 
tion could depend upon a viral infection as in 
hepatitis, but in other situations no adequate ex- 
planation is available. The rationale for the effect 
of 6-TG, at present, is that it suppresses protein 
biosynthesis in cells which form antibody globu- 
lins, thus preventing damage to host tissue from 
antigen-antibody combination. The findings in 
patients and in the rabbit studies are consistent 
with this concept, although possible anti-inflam- 
matory effects of 6-TG are also being investigated. 

From the limited studies now available it is 
difficult to predict the benefits of this new thera- 
peutic approach to a group of diseases previously 
treated with only modest success. The tendency 
to relapse already evident in some cases and the 
pronounced hematologic and probably other toxic 
effects suggest some limits to the usefulness of the 
present agents. Nevertheless, two important 
points are already clear; first, a new principle 
of therapy has been demonstrated which is worthy 
of a careful follow-up; secondly, the fact that 
remissions can be achieved with antimetabolite ad- 
ministration in diseases of unknown eitiology 
opens a new avenue for investigation of the patho- 
genesis of these diseases. Both points are under 
further study. 



208 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Respiratory Viral Agents in Normal Volunteers 

The use of prisoner volunteers in the study of 
viral respiratory agents was begun 2y 2 years ago. 
More than 500 men have come to the Clinical 
Center for these studies, 241 of them this year. 
One of the recent developments of the program 
has been the administration of a respiratory viral 
agent in a submicron size aerosol. This has per- 
mitted a rather precise estimate of the small dose 
of virus required to produce virus infection, ill- 
ness, and antibody response. It has allowed com- 
parison of the characteristics of disease produced 
by lower pulmonary tract inoculation with that of 
the upper tract, and is now leading to studies di- 
rected at answering the question of the mechanism 
of natural air-borne spread of respiratory viral 
disease. 

In the studies undertaken with the staff of the 
Biological Laboratories at Fort Detrick, Coxsackie 
A-21 (Coe agent) has been found to produce dis- 
ease (ID 50 ) with submicron size aerosol particles 
with 28 TCID 50 per volunteer in a 10 liter inhala- 
tion. The 95 percent confidence limits are 16 to 
63 TCID S0 . The smallness of this dose is empha- 
sized when it is recalled that only about 50 percent 
of the dose is retained. 

The equipment for this work was developed at 
Fort Detrick. Inoculation is quickly and simply 
performed in a truck semitrailer which contains 
all the necessary equipment and is brought to the 
Clinical Center for this purpose. It is apparent 
that this same equipment could be modified easily 
to provide rapid and accurate inhalation of viral 
vaccines, and in view of the stability of small par- 
ticle aerosol clouds, it is apparent that larger scale 
inoculation would require only modification of the 
equipment along lines already well-understood. 
Part of the future effort in this program will be 
concerned with vaccine development. 

Anti-Viral Therapy 

In what is hoped may become a larger effort 
three antiviral substances have been tested in vol- 
unteers this year. The agent reported by Kauf- 
man to be effective in herpetic conjunctivitis, 5- 
iododeoxyuridine, was found to be ineffective in 
conjunctivitis in volunteers caused by adenovirus 
type 27. A thiopyrimidine compound, active 
against influenza in mice, was found to be ineffec- 



tive in treatment of induced human Asian (strain 
305) influenza. More recently an irradiated prep- 
aration influenza virus (PR-8) which stimulated 
interferon production in tissue culture and in mice 
sufficient to protect against influenza and other 
viral infections, is being tested in volunteers. This 
program has moved slowly because less than 5 
percent of prisoner volunteers are free of antibody 
to Asian influenza virus and getting sufficient 
numbers of such candidates for study is difficult. 

Malaria 

During the year the study of malaria antibody 
formation was continued. Studies in volunteers 
revealed that antibody to the simian strain, Plas- 
modium cynomologl ( B ) , reacts to a greater extent 
with this parasite than with two human vivax 
strains (Chesson and Venezuelan). Antibody to 
the vivax strains reacts with them and to the 
simian parasite to the same degree. These find- 
ings were interpreted to indicate that the simian 
parasite possesses antigens in common with human 
vivax strains, but in addition, has an antigen dis- 
tinct from the vivax plasmodia. 

Study of sera obtained last year from Ghana 
has revealed a high titer of antibody when P. fal- 
ciparum is used in a test, but low titers when vivax 
or the simian plasmodia were used. This evidence 
of specificity of the test for malaria antibody sug- 
gests a great area of usefulness of the procedure. 

Biochemical Assay for Penicillin 

Despite the many years of intensive investiga- 
tion of pencillin derivatives no satisfactory bio- 
chemical assay for these compounds has been 
developed. In recent months, in the course of an 
investigation of penicillin allergy, an acid break- 
down product of penicillin, penicillenic acid, was 
found to be stabilized by the addition of a mer- 
curic salt, and could be quantitatively measured 
by UV spectrophotometry. This permitted an 
accurate assay of most penicillin derivatives on 
the market down to concentrations as low as 2 meg 
per ml. It was further found that the reaction 
was less variable in the presence of albumen or 
serum. The test could be completed in about one 
hour, a much shorter time than with the bioassay 
using; Sarcina lutea. In tests of unknown sera 
there was no significant difference in results by 
biochemical and bioassay. This test is therefore 






NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



209 



applicable to the assay of biologic fluids and can 
be conveniently used as an adjunct in pencillin 
therapy. 

Fungus Disease 

This program continues to work toward im- 
provement in the diagnosis and treatment of sys- 
temic fungus infections. Clinical reports are pub- 
lished regularly and serve to alert practicing phy- 
sicians to attempt to find new cases. Such efforts 
benefit the program, since it stimulates referral of 
cases here for study. From such referrals, 67 
patients were selected for study this year. 

As experience accumulates new clinical syn- 
dromes are discovered. The report this year of 
five adults stricken with histoplasma meningitis is 
the first description of the disease diagnosed in 
adults prior to autopsy. Only one of the five pa- 
tients died. 

From the group of patients with cryptococcal 
meningitis, the largest group ever assembled in one 
institution, has come an encouraging report of the 
efficacy of amphotericin treatment. Of 30 pa- 
tients with this usually fatal illness, only 5 did not 
respond to treatment. All five had some other 
serious disease, usually cancer or diabetes mellitus. 

Plasma Cell Tumors and Antibody Production 

Using ovalbumin as an antigen it was shown 
that mice with implanted plasma cell tumors did 
not respond to primary stimulation with ovalbu- 
min. The antigen was given near the time of 
tumor implantation. Approximately 10 days 
later, however, when tumor growth was proceed- 
ing rapidly, response to a second dose of ovalbu- 
min was followed by significant antibody response. 
It was the interpretation that tumor growth in 
some manner prevented development of primary 
response, but despite increasing tumor growth, the 
additional stimulation of a second dose of antigen 
was sufficient to cause antibody production in the 
few remaining antibody-producing cells of the 
mouse. It was felt that the tumor prevented pri- 
mary response but that tumor cells did not partici- 
pate in the production of antibody. These obser- 
vations may have some bearing on mechanisms of 
immunity in humans with plasma cell and other 
diffuse tumors. 



LABORATORY OF PARASITIC DISEASES 

The work of this Laboratory has encompassed a 
broad range of subjects and approaches to para- 
sitic disease problems. 

Angiostrongylus Cantonensis 

Quantitative experimental infections with 
Angiostrongylus cantonensis in rats and monkeys 
have revealed pathologic changes consistent with 
the clinical disease syndrome eosinophilic menin- 
gitis. Work with A. cantonensis has also shown 
that this worm undergoes larval development in a 
large variety of freshwater snails as well as slugs, 
and that larval stages can survive in both fresh 
and salt water. These findings suggest that the 
worm may be able to reside in various paratenic 
hosts (secondary carriers in which no development 
occurs). This could explain the epidemiological 
relation between the ingestion of raw fish and the 
occurrence of eosinophilic meningitis, suspected as 
due to A . cantonensis, in Tahiti. 

Adults of A. cantonensis have been maintained 
in vitro for 80 days and metabolic products de- 
rived from these cultures have proven useful as 
antigens in a hemagglutination test on the sera of 
some rats. These antigens have come only from 
incubates of female worms during eai'ly cultiva- 
tion, when they are still depositing eggs. 

Experimental Schistosomiasis 

Work on the pathophysiology of experimental 
schistosomiasis revealed shifts of worms to the 
lungs on treatment of mice that had developed 
portal-systemic collateral circulation. Repeated 
infection and repeated treatment of mice, allowing 
delivery of large numbers of eggs and worms to 
the liver, did not change the pathologic picture of 
the infection; and the mice rapidly returned to 
normal as judged by the physiologic and anatomic 
criteria. Treated mice showed no resistance to 
reinfection, except slightly slower development of 
worms. These results are of interest in relation 
to the management of human infections. 

Snail Hosts of Schistosomes 

Study of the biology and control of snail hosts 
of schistosomes has shown that two species of 



210 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Acanthamoeba-like protozoa parasitize tissues of 
snails. These organisms do extensive tissue dam- 
age but do not, apparently, decimate snail popula- 
tions. However, they may be important in re- 
stricting populations, and adverse effects on the 
amoebae of some biocides could, conceivably, re- 
sult in later increase in snail densities. Observa- 
tions on seasonal changes in the apertural lamellae 
of Australorbis glabratus, and their relation to 
migration of snails out of water are important in 
understanding the ability of snails to survive dry 
seasons in some areas. 

Antimony Compounds and Diet in Treatment 

The efficacy of antimonial compounds against 
Schistosoma mansoni has been shown to be influ- 
enced by such dietary factors as acid-salt residues, 
Mg-Ca rations, and amount of crude fiber. How- 
ever, these factors account for only a fraction of 
the enhanced (up to 30-fold) drug activity seen in 
mice on a purified semisynthetic diet. Antimony 
compounds given either by oral or parenteral 
routes have increased activity in such mice. These 
findings have considerable significance in relation 
to the chemotherapy of schistosomiasis in develop- 
ing countries, where the diet of the people may be 
a major factor in failure to obtain good results on 
treatment. The findings that purified low residue 
diets induce self-cure of mice with various nema- 
tode infections, also have bearing on the manage- 
ment and control of intestinal helminthiases in 
many parts of the world. Aside from the practical 
aspects, these data provide a stimulus for basic 
work on the factors of toxicity, immunity, etc., 
that may be operative. 

Axenic Cultivation of Amoebae 

Work on the axenic cultivation of Entamoeba 
histolytica has been continued, with three strains 
now established in, and a fourth strain being ac- 
climated to, axenic conditions. Noteworthy is the 
observation that the more hypotonic the overlay 
of the medium, the better its ability to support 
initial axenic growth. This suggests that the nu- 
trition of amoebae is related to physical factors 
in the medium, stimulating pinocytosis. Chick 
embryo extracts of various types are still needed 
to support initial axenic growth, but once estab- 
lished, all strains can be adapted to growth with- 
out them. Substitution of a liver extract for 
yeast extract in the maintenance medium has in- 



creased amoeba yields. Trichomonas tenax has 
also been grown in axenic culture for the first time. 






Biochemistry of Parasites 

Continuing studies on the biochemistry of proto 
zoa has added four enzymes to our catalog of those 
known from Trichomonas vaginalis and increased 
our knowledge of purification procedures in their 
study. 

Further work on the absorption and excretion of 
sugars by Taenia taeniae formis has shown that the 
uptake of glucose from the medium by adult worms 
is essentially independent of the glucose concen- 
tration, while on the other hand, more galactose 
is consumed at higher concentrations. Glucose is 
secreted by both larval and adult worms when kept 
in sugar-free Tyrode's solution ; in the presence of 
low glucose concentrations, adults begin to absorb 
the sugar while larvae continue to excrete it until 
the concentration is eight times higher than the 
threshold for adults. The presence of other sugars 
appears to have little effect on larval glucose ex- 
cretion. The lack of dependence on concentration 
of glucose absorption by adults suggests an active 
transport mechanism. 

The activation of succinate oxidation by ATP, 
reported last year, appears to occur at the level of 
dehydrogenases, rather than in the respiratory 
chain. Uncouplers of oxidative phosphorylation, 
such as the halophenols, are assumed to be bound 
to mitochondrial lipids. However, work on lipid- 
depleted mitochrondria showed binding of penta- 
chlorophenol (PCP) to the same extent as in lipid- 
rich controls. Furthermore, studies with plasma 
albumin, which reversed the uncoupling effect of 
PCP indicate that protein binding may be an im- 
portant factor in the action of uncoupling agents. 
These findings are of general biochemical interest 
in regard to the theory of respiratory chain 
phosphory 1 ation ^ 

Toxoplasmosis 

Research on toxoplasmosis has revealed cysts in 
the lung and intestine of mice. From these sites 
they can be shed to the outside environment. 
Studies on newborn mice from chronically infected 
mothers indicate that either immune paralysis or 
tolerance may develop when congenital transmis- 
sion occurs during chronic infection. The finding 
of a toxin in the course of the serological studies 
is noteworthy. This toxin is entirely different from 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



211 



that reported previously, about which there is con- 
siderable doubt, and is similar to sarcocystin or to 
that from coccidia. Results with the hemagglu- 
tination test have been so consistent that it is now 
adopted for routine diagnostic use. 

LABORATORY OF BACTERIAL DISEASES 

There has been a significant change in the re- 
search program of the Laboratory of Bacterial 
Diseases during the past year. 

Pleuropneumonia-Like Organisms 

Because of the growing importance of the 
pleuropneumonia-like organisms in human dis- 
ease, the real need for basic studies on this group 
of organisms, and the long-standing interest of the 
Laboratory in the PPLO and the L-forms of bac- 
teria, a new research program of basic studies on 
the PPLO and the L-forms of bacteria has been 
initiated. 

To date, considerable quantitative growth data 
on representative strains has been accumulated, 
and some methods of preparation of antigens and 
antisera have been explored. Of particular inter- 
est are preliminary results of the use of certain 
cell fractions as erythrocyte sensitizing antigens. 
If such antigens can be developed in quantity, 
quantitation of immune responses may be made 
much more sensitive and accurate. 

Intracellular Parasitism 

The studies on the intracellular parasitism proj- 
ect have continued in the same general area as pre- 
viously, that is, on antibody production in vitro of 
cells removed from immunized animals. Com- 
parative studies have been done on cells from the 
spleen, lung, and peritoneal cavity of guinea pigs. 
No new antibody production was detected in 
macrophages perfused from the lung. Cells from 
the peritoneal cavity yield better results in our ex- 
periments than those from the spleen. The cell- 
type producing antibody is still uncertain. These 
studies are being continued. 

LABORATORY OF PARASITE CHEMO- 
THERAPY 

The main research effort continued to be centered 
on overall problems in malaria. However, investi- 
gations involving parasitic infections in general, 



with special emphasis on schistosomiasis, intesti- 
nal parasites and viruses were pursued. 

Malaria — Human 

The prophylactic effect of a new antimalarial 
drug appears promising. Twenty-two of twenty- 
four volunteers given a single intramuscular injec- 
tion of CI-501, 5 mg/kg, and challenged by bites 
of infected mosquitoes on days 5 to 322 have been 
protected against vivax malaria for from 198 to 
357 days. Two volunteers became infected after 
169 days and 341 days following mosquito bites 
on days 167 and 322. Four volunteers given a 
single intramuscular injection of the same com- 
pound, CI-501, and challenged on days 9 to 12 with 
the Southern Rhodesian strain of Plasmodium 
falciparum, by blood inoculation, became infected. 
Four volunteers challenged on day 5 (after drug 
injected) by mosquito bites were protected. The 
Southern Rhodesian strain is not fully sensitive to 
chlorguanide and may be resistant to the triazine. 
Two volunteers challenged with the McLendon 
strain by blood inoculation on days and 5 were 
protected. 

A number of malaria strains exhibit resistance 
to chloroquine, the most important of modern anti- 
malarials. The Colombia strain of P. falciparum 
was infective for mosquitoes for extended periods 
after chloroquine treatment which would cure a 
normally sensitive strain. The Thailand strain of 
P. falciparum was resistant to chloroquine, 
amodiaquine, pyrimethamine and chlorguanide 
but sensitive to the suppressive effect of quinine 
and to the sporontocidal effect of primaquine. 
Two cases of falciparum malaria acquired in 
Brazil were resistant to several normal courses 
of therapy with chloroquine. One patient was 
cured by higher doses of chloroquine and one was 
cured by a 10-day course of quinine totalling 
10 gm. 

Malaria — Simian 

Continued studies of the susceptibility of man to 
monkey malaria have added important informa- 
tion. Serial passages of the M strain of Plasmo- 
dium cynomolgi by blood inoculation have been 
made in volunteers with a maximum of eight pas- 
sages in one line. The peak parasitemia was not 
strongly correlated with passage number but the 
highest peak parasitemia (7,200 per cmm) was 
found in the eighth passage. In a smaller series 



705-685 — 63- 



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212 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of subinoculations of the B strain the highest peak 
parasitemia was 1,150 per cmm. None of nine vol- 
unteers infected by mosquito bite relapsed after 
treatment of their primary attacks with quinine. 
One of three volunteers bitten by a single infected 
mosquito became infected and remained positive 
for 17 days. 

Two of three volunteers were infected, by mos- 
quito bite, with a strain of P. cynomolgi recently 
isolated from a Malayan pigtail monkey. The in- 
fections were benign and parasite counts did not 
exceed 50 per cmm. 

One of two volunteers bitten by Anopheles free- 
borni infected with Plasmodium brasilianum be- 
came positive in 63 days. This is the first known 
infection of man by a New World simian malaria. 

Two new species, Plasmodium coatneyi of 
Macaca irus and a tertian parasite of the white- 
handed gibbon were described. The former is a 
tertian parasite with asexual stages like P. falci- 
parum. The latter resembles Plasmodium vivax 
but does not cause enlargement of erythrocytes. 
An additional new parasite of a non-simian host, 
the Malayan flying lemur, also has been described. 

Infected salivary glands of Anopheles freeborni 
injected into a rhesus monkey produced an infec- 
tion of P. coatneyi which was positive on the 15th 
day. Anopheles quadrimaculatus, A. crucians and 
A. walkerh were not susceptible to P. coatneyi. 
Anopheles freeborni was susceptible to P. fieldi 
but Anopheles quadrimaculatus was not. Anoph- 
eles freeborni and Anopheles quadrimaculatus 
were susceptible to both P. gonderi and P. inui. 

Biochemical Studies 

Although Plasmodium berghei is susceptible to 
folic acid antagonists (aminopterin, amethopterin 
and pyrimethamine) PABA is a complete substi- 
tute for folic acid while folic acid is only a partial 
substitute for PABA. Moreover deficiency of 
folic acid in animals supplied with adequate 
PABA favors rather than inhibits the parasites. 
A pyrimethamine resistant strain of P. berghei 
was found to have an increased need for PABA 
and an increased sensitivity to sulfonamide. 
These results are all consistent with the hypoth- 
esis that the parasite uses PABA to f orm a sub- 
stance similar to but not identical with folic acid 
and that resistance to pyrimethamine involves a 
change in the nature of the folic acid analogue uti- 
lized in the metabolic system of the parasite. 



Pantothenic acid deficiency as well as panto- 
thenic acid analogues favor rather than inhibit 
P. berghei. The combination of deficiency plus 
antimetabolite has an antimalarial effect. These 
results are interpreted as meaning that the parasite 
needs pantothenic acid but is less sensitive to 
deficiency than the host. The more complete de- 
privation produced by the combined effect of 
dietary deficiency and antimetabolite is sufficient 
to inhibit the parasite. 

The ability of malarial pigment to form a com- 
plex with chloroquine was predicted from theory 
and confirmed by both chemical methods and bio- 
assay. The failure of chloroquine to inhibit the 
incorporation of labelled amino acids and the 
affinity of hematin for chloroquine suggest that 
the mode of action of chloroquine may involve 
degradation of hemoglobin rather than a synthetic 
process. 

Immunological Studies 

Fluorescent antibody techniques have revealed 
that antibodies to Plasmodium malariae appeared 
between the 15th and 28th day after inoculation 
and persisted until the 120th day. Antibodies to 
P. falciparum persisted up to 18 months but with 
a decline in titer by the 12th month. Cross reac- 
tions between P. malariae and P. falciparum 
occurred. 

Far East Research Project Studies in Malaya 

Four species of simian malaria including one 
new to science (P. coatneyi) have been isolated 
from wild-caught specimens of Anopheles hackeri 
by injection of sporozoites into uninfected mon- 
keys. 

A wild specimen of Anopheles leucosphyrus 
infected with P. inui was captured in the act of 
biting man. Also it was proved that Anopheles 
balabacensis introlatus is a vector of monkey 
malaria and other studies have shown that this 
species too attacks man about as readily as it 
attacks monkeys. An area in Cambodia was lo- 
cated where A. balabacensis balabacensis was com- 
mon. The attack rate on monkeys in the canopy 
and man on the ground was nearly equivalent. 
Infected mosquitoes were found and identification 
studies of the sporozoites are underway. 

Mansonia uniformis was consistently susceptible 
to experimental infection with two strains of P. 
cynomolgi, but sporozoites did not regularly ap- 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



213 



pear in the salivary glands and attempts to infect 
monkeys by injection of sporozoites from oocysts 
have failed. Other susceptibility studies with over 
20 species of Malayan Anopheles showed that 
Anopheles barbirostris, A. umbrosus, and A. hyr- 
canus were not very susceptible to P. cynomolgi 
bastianellu while A. macidatns, A. kochi and A. 
sundaicus were very susceptible. A. bcdabacensis 
introlatus was highly susceptible to P. cynomolgi 
but practically insusceptible to P. c. bastianellii. 
About 40 inoculations of blood from aborigines 
with malaria into uninfected monkeys have not 
revealed strains that would grow in monkeys. 

Schistosomiasis 

Three of 104 compounds tested against Schis- 
tosoma mansoni were active. Two were less active 
derivatives of Miracil D and one was a confirma- 
tory test of a compound produced by Abbott. 
Among the inactive compounds were 17 enzymes, 
mostly proteolytic. 

Virus-Mosquito Larvae Associations 

Larvae of Aedes aegypti and Anopheles albi- 
manus mosquitoes acquired Semliki Forest virus 
from an experimentally contaminated aquatic en- 
vironment. The resulting infections in adult 
mosquitoes were of low titer and prevalence. 

Intestinal Parasites 

Stilbazium iodide 10 mg/kg eliminated Ente- 
robiics vermicularis infection in children and 
Strongyloses stercoralis in adults. It was mod- 
erately effective against hookworm and Trichwris. 
Bephenium gave excellent results against hook- 
worm and dithiazanine was highly effective 
against Trichwris. 

LABORATORY OF INFECTIOUS DISEASES 

This group of investigators, one of the largest 
in the Institute, pursued important studies on a 
variety of infectious processes. These ranged 
from the role of viruses in cancer, the viral etiol- 
ogy of respiratory disease, and antibacterial ef- 
fects of sea water to the ecology, immunology and 
experimental treatment of fungus diseases. 

Adenovirus Types 12 and 18 as Cancer Viruses 

Trentin's discovery that type 12 adenovirus 
caused cancers in hamsters was promptly con- 



firmed and his findings extended as follows: (a) 
Four separate freshly isolated strains of adeno- 
virus 12 recovered from anal swab specimens of 
4 Washington, D.C.. infants readily produced 
similar cancers in animals; (b) The oncogenic 
activity was shown to be associated with replica- 
tion of a particle inducing a unique specific com- 
plement-fixing antibody to adenovirus 12, but only 
in those hamsters developing cancer; (c) type 18 
adenovirus, which share a number of unique prop- 
erties with type 12, also produced similar cancers ; 
(d) similar fatal but apparently nonmetastasiz- 
ing fibrosarcomas were produced locally within 40 
days following intraperitoneal, intracerabral, 
subcutaneous and intrathoracic injections. 

Twenty-six other adenoviruses have not pro- 
duced cancers in hamsters during 6 or 9 months' 
observation. Children infected with adenoviruses 
12 and 18 are being followed and studies of the 
mechanism of adenovirus oncogenesis in animals 
more recent efforts. 

Papilloma Viruses 

Common complement-fixing antigens were 
found in bovine, canine and rabbit papillomas. 
These unsuspected relationships suggest a search 
for a similar antigen in human papilloma viruses. 
Interestingly enough, antibody reactions to ani- 
mal papillomas can be demonstrated in human 
serums. Bovine papillomas were found to pro- 
duce subcutaneous fibromas in hamsters and ap- 
parently a specific complement-fixing antibody 
response as well. 

Natural History of Polyoma Virus 

The natural cycle of polyoma virus was demon- 
strated previously in rural (agricultural) ecolo- 
gies. Grain storage areas on farms and mills pro- 
vide stable environments suitable for harboring 
large and dense populations of Mus muscuhis and 
numerous effective portable sources of polyoma in- 
fection. Similar results were obtained in surveys 
of grain mills in Indiana, Georgia and Florida. 
Polyoma was isolated from 49 of 109 grain speci- 
mens. A number of other virus infections were 
detected in mice in all these areas. Their impor- 
tance to human and animal health is unknown, but 
two of them, reoviruses and lymphocytic meningi- 
tis (LCM), are known to infect man and domestic 
animals. 






214 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Respiratory Virus and Vaccine Studies 

Much of the current activities of the respiratory 
program are now directed toward development of 
effective respiratory disease vaccines. Since the 
programs now underway involved setting up new 
commercial contracts during 1962, there is little 
that is new to report as yet. However, important 
new observation were made on certain respiratory 
viruses having direct importance to the vaccine 
effort. Volunteers were infected when fed adeno- 
viruses 4 or 7 in enteric coated capsules. Selective 
infection of the intestinal tract, while bypassing 
the respiratory tract, provides a sound basis for 
pursuing live adenovirus immunization. Intro- 
duction of live virus by an atypical route may rep- 
resent a solution to the thorny problem of adeno- 
virus immunoprophylaxis. At present, effective 
safe adenovirus vaccines are not available because 
of difficulties in producing potent lots of adeno- 
virus material free of simian virus (SV-40). 

In other studies naturally occurring reinfection 
with RS virus was demonstrated in adults. This 
is an important finding in the natural history of 
RS infection. It also poses a problem regarding 
vaccination of adults, since upper respiratory ill- 
ness can occur despite the presence of high levels 
of neutralizing antibody. 

Complexity of the Rhinoviruses 

Workers in LID, along with scientists in other 
laboratories, have identified at least 40 different 
serotypes and the end is not in sight. Before a 
rational approach to vaccination can be formu- 
lated, the rhmovirus group must be completely de- 
fined and the importance of the various virus sero- 
types established. A preliminary analysis of our 
volunteer data suggests that neutralizing antibody 
may be only moderately effective in preventing 
rhinovirus disease. The findings also suggest that 
reinfection may also be of importance in the natu- 
ral history of the rhinoviruses. 

Mycoplasma Agents in Respiratory Disease 

The Eaton agent has now been clearly estab- 
lished as a mycoplasma (pleuropneumonia-like or- 
ganisms) instead of a virus, and for the first time 
accurate, easily performed diagnostic tests for 
Eaton agent pneumonias have been developed. 
This represents the first association of a myco- 
plasma with human respiratory disease; in fact, 
with any type of illness in man. New techniques 



for recovery of Eaton agent on artificial media 
have provided simple methods for isolating th( 
PPLO from infected individuals. This should fa- 
cilitate future diagnostic and epidemiologic stud- 
ies. Growth of the organism in broth suspension 
has made possible the preparation of a specific 
complement-fixing antigen, thus simplifying tht 
problem of serodiagnosis. In addition, it provides 
the basis for growing the organism in bulk culture 
for vaccine production. It was found that a solu- 
ble hemolysin of Mycoplasma pneumoniae (Eaton 
agent) completely lyses guinea pig red cells, where- 
as this property is not possessed by other human 
PPLO. The implications of this finding to quan- 
titation of the organism and to identification of 
field isolates are obvious. 

In collaboration with the Laboratory of Clini- 
cal Investigations, infection of antibody- free vol- 
unteers with high passage artificially propagated 
M. pneumoniae was undertaken. Evidence of at- 
tenuation was obtained in this study. There were 
no illnesses among the 12 volunteers thus far in- 
fected indicating evidence of attenuation of the 
strain. These findings provide a basis for pur- 
suing the development of an attenuated live myco- 
plasma vaccine. 

Virus Infections and Human Cancer 

A serological survey for viral infections in leu- 
kemia patients and closely matched nonleukemic 
controls, in St. Louis Hospital, Paris, France, re- 
vealed evidence of numerous virus infections. Al- 
though differences in virus experiences were ob- 
served in the two groups, the significance and 
possible explanation for these differences require 
further study. 

Cooperating with the Laboratory of Clinical In- 
vestigations, NCI, and the Human Cancer Virus 
Task Force, comprehensive collections of serum 
specimens from patients with all types of cancer 
were begun. A serum center similar to that set up 
with NINDB for the study of birth defects is 
planned in conjunction with the new cancer- virus 
facilities proposed for our collaborative studies 
with NCI. 

Cancer Viruses in Vaccines and Foodstuffs 

In 1959, it was pointed out that studies on poly- 
oma and extraneous viruses in cell systems and 
natural ecologies held serious implications for 
viral vaccines, particularly live vaccines. The 



NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 



215 



simian SV-40 virus was reported by others in 
poliovirus vaccines in 1961. Subsequently, its on- 
cogenic behavior in hamsters and in cultures of 
human cells was demonstrated. In 1961, wide- 
spread contamination of cereal grains used for 
human as well as domestic animal nutrition with 
polyoma and other viruses of the house mouse was 
reported. 

Recently, the frequent presence of chicken lym- 
phomatosis virus in fertile eggs was reported. 
This has caused a "background noise" problem 
in chick embryo virus study systems. It also poses 
a problem to those concerned with the safety of 
virus vaccines made in chick embryo tissues and 
with the purity of widely used food staples. 

Pacific Research Laboratory, Hawaii 

In the summer of 1962, a new laboratory, the 
Pacific Research Section, was established in Hono- 
lulu, Hawaii, as a separate section of LID for 
the primary purpose of studying eosinophilic 
meningitis in the Pacific area. Further evidence 
that the widespread occurrence of eosinophilic 
meningitis in the Pacific area (especially in 
French Polynesia) may be due to Angiostrongylus 
cantonensis, a parasite of rats, was acquired by 
demonstrating the ability of larval forms to invade 
the brains of rhesus monkeys. Cases continued to 
occur in French Polynesia during 1962. 

Rubella Virus Isolation in Tissue Cultures 

Collaborative work with NINDB not only con- 
firmed preliminary observations of the Walter 
Reed Army Institute of Research group on the 
isolation of rubella virus, but helped establish the 
viruses as the cause of rubella. The latter was 
achieved by sero-epidemiological studies and by 
the induction of rubella in volunteers shown by 
serological tests to be susceptible before inocula- 
tion. Immune volunteers did not develop the 
disease. Very recent preliminary results suggest 
that inactivitated rubella vaccines will present the 
experimental induction of rubella in susceptible 
volunteers. 

Enterovirus Studies 

The properties and natural behavior of enter- 
oviruses received considerable investigation in 
1962. ECHO virus type II was shown in Junior 
Village to be associated with a diarrheal illness 
in infants. A number of enteroviruses were shown 



to contain hemagglutinins which greatly simpli- 
fied their typing and classification. Five newly 
recognized viruses (with entero-rhinovirus prop- 
erties) were characterized and submitted for clas- 
sification to the American Enterovirus Committee. 
Fifty more strains of reovirus were identified 
from feces of cattle ; most were types as reovirus 
types 1 and 3. Inoculation of human volunteers 
with bovine reoviruses produced infection, but no 
recognizable disease. 

Anti-Bacterial Effect of Sea Water 

In the attempt to identify the macromolecule 
in sea water which is lethal for staphylococci, it 
was found that the activity is lipase-sensitive. 
This suggests that a lipid is the antibioticlike com- 
pound. This, except for a few steroids, is the first 
time this class of compounds has been implicated 
in such activity. 

Studies on Hydrogenomonas 

The electron-transport system of Hydrogeno- 
monas has been broken down further into its com- 
ponent parts and it is now known that, following 
the reduction of DPN by H 2 , the reduced nucleo- 
tide is oxidized via a flavin, vit K 3 (menadione) 
and a cytochrome. Some purification of mena- 
dione reductase has been achieved. It is empha- 
sized that all of these enzymes in the complex are 
soluble, thus making investigation of the linkage 
between oxidation and phosphorylation more 
amenable to study. 

Siderophilin 

Carbon dioxide has been found to be essential 
for the formation of the siderophilin-iron chela- 
tors and indications are that only the 2 Fe :1 sider- 
ophilin complex is formed. Improved methods of 
purification of biologically active siderophilin 
have been devised and using such preparations, 
further observations have been made on the time 
sequence of the uptake of Fe by reticulocytes fol- 
lowing erythropoietic stimulation. A new immu- 
nological method for the determination of small 
amounts of siderophilin has been worked out. 

Bacteriophage Treatment for Urinary Infections 

Studies on the utilization of specific bacterio- 
phage for the treatment of urinary tract infec- 
tions refractory to antibiotic therapy have been 
continued. Of clinical significance is the fact that, 



216 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



following phage treatment, the urine was initially 
cleared of an antibiotic-resistant pathogen. After 
as much as 30 days later, baccilluria re-occurred, 
this time with a different pathogen, but now, in a 
high percentage of cases, the new offending cell 
was sensitive to those antibiotics initially found 
to be ineffective. 

Detoxification 

Detoxification studies have progressed further 
on potential tuberculostatic, fungistatic, parasiti- 
cidal, viricidal and antitumor agents. The anti- 
viral activity extracted from various molluscs has 
been characterized as an acetylneuraminic acid- 
glycoprotein composed of sugars, sialic acid and 
18 amino acids. Pyridyl- and guanyl-hydrozones 
have been tested for antitumor activity and have 
passed various preliminary tests carried out by 
the Cancer Chemotherapy Screening Center. 

Cell Wall Formation 

By the use of the immunofluorescence technique, 
it has been found that new cell wall is laid down 
in a diffuse, intercalated manner in the gram- 
negative Salmonella typhosa as opposed to the 
polarized, sharply demarcated partitioning in the 
gram-positive Streptococcus pyogenes. The same 
techniques have been used to follow formation of 
protoplasts induced by penicillin in S. typhosa. 
Fine structure of walls, membrane and protoplasm 
are being studied by electron microscopy u